The syncytial cell Physarum polycephalum exhibits cytosolic streaming, cell migration, and cell network remodeling within a single cell. It is unclear how cytoskeletal elements are spatiotemporally organized to mediate these processes. Here, we combine live and fixed imaging of Physarum F-actin to uncover organizational and dynamic signatures of the actin cytoskeleton across different cell structures. We find circumferential actin filaments constrict and relax to drive peristaltic contractions in feeding tubes, while migratory fans are characterized by actin patches and comets. Our work presents previously undescribed actin structures in Physarum and demonstrates how different actin-based behaviors coexist within a syncytial cytoplasm.
","acknowledgements":"This work was performed at the 2023 Physiology course at the Marine Biological Laboratory (MBL), Woods Hole, directed by Dr. Nicole King and Dr. Daniel Fletcher, and in the lab of Dr. Amy Gladfelter at Duke University. We thank NIH and MBL for funding and supporting the course that made the work possible, as well as the post-course research. The course was funded by NSF grant #2014108 and Burroughs Wellcome Fund Award # 1282312, We thank Nikon and Olympus for contributing systems to the MBL Physiology course. We thank Rui Jiang and Isaac Breinyn, students of the 2023 MBL Physiology course, for capturing additional movies of Physarum polycephalum that are not included in this manuscript. We also thank the 2023 MBL Physiology course for funding the post-course research of A.C. and Z.G. We thank Dr. Christine Field and Dr. Tim Mitchison for providing LifeAct-GFP protein for injections.
","authors":[{"affiliations":["Columbia University","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Biomedical Engineering","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"adc2174@columbia.edu","firstName":"Andrew","lastName":"Countryman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","writing_originalDraft","writing_reviewEditing","visualization"],"email":"zmg2@duke.edu","firstName":"Zachary","lastName":"Geisterfer","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["supervision","conceptualization","writing_reviewEditing"],"email":"amy.gladfelter@duke.edu","firstName":"Amy","lastName":"Gladfelter","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Description: Time-lapse movies of Physarum actin dynamics in tubes (left) and fans (right), as visualized with LifeAct-GFP. The scale bar and time stamps are shared across the two movies.
","doi":null,"resourceType":"Audiovisual","name":"supplementary movie.mp4","url":"https://portal.micropublication.org/uploads/797cd009abcaee7acdd12741de1b62c2.mp4"}],"funding":"The Physiology course was funded by NSF grant # 2014108 and the Burroughs Wellcome Fund Award # 1282312. Z.G is supported by NIH grant # 1F32GM151858-01.
","image":{"url":"https://portal.micropublication.org/uploads/db470c8fe5e8dfe00b749b77e45c5911.png"},"imageCaption":"(A) Brightfield image of the macroplasmodium of Physarum polycephalum, with the feeding tubes (blue box) and the migratory fan (red box) marked. Scale bar = 1 mm. (B) Brightfield images with inverted LUT showing the expansion of a feeding tube during peristaltic contractions. Scale bar = 50 µm. (C) Fixed, phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (D) Phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). (E) Actin assemblies visualized through LifeAct-GFP in the feeding tube. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (F) Actin structures, labeled with LifeAct-GFP, in a feeding fan. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm.
","imageTitle":"Actin ultrastructure varies across a single Physarum polycephalum macroplasmodium
","methods":"Physarum polycephalum macroplasmodia were grown on 1.7% (wt/vol) low-fluorescence water agar and fed oat flakes every two days. Twenty-four hours before imaging, 1 cm2 agar blocks containing a region of the cell were transferred to a new agar plate and left to develop a mature network in the absence of oat flakes. After starving the sample for 24 hours to reduce autofluorescence, the samples were prepared for live or fixed imaging.
For fixed imaging, sections of the network were prepared by excising an agar block containing structures of interest and gently immersing the entire agar block and specimen into 4% formaldehyde for 10-15 min at RT. After fixation, samples were rinsed three separate times using RT PBS buffer. Each rinse was approximately the volume of the initial formaldehyde immersion and was left for 5 min to allow buffer exchange within the agar block. After rinsing the specimen, samples were immersed in PBS buffer containing 660 nM Phalloidin-Rhodamine for 10 min at RT. The specimen was rinsed once in PBS as described above, and subsequently all buffer was removed from the imaging well containing the sample. 3D fluorescence microscopy was performed on a Nikon Ti2-E inverted microscope equipped with a Yokogawa CSU-W1 spinning disk confocal. Images were acquired using a 40x Plan Apo silicon immersion objective (NA = 1.25). Selected images were displayed as a z-depth colored intensity projection for Figure 1C,D.
For live samples, agar blocks containing regions of interest were transferred to a stereomicroscope (Zeiss Stemi 305) equipped with a FemtoJet 4i microinjector (Eppendorf). Injection needles (World Precision instruments; OD=1.0mm, ID=0.75mm) were pre-loaded with ~3ul of LifeAct-GFP (250 µM) and set to a shallow angle of 5-10° relative to the face of the agar block. The compensation pressure was set to 60 hPa, the injection time was set to 0.5 seconds, and the injection pressure was initially set to 80 hPa. The needle was delicately introduced to a larger tube section of the specimen where the LifeAct-GFP was released. Due to the large intracellular pressure variation within and between Physarum samples, the injection pressure was increased in intervals of 10 hPa until injection resulted in visible release of LifeAct-GFP into the cell. Then, the injection was sustained until reaching a final injection volume of approximately 200 nL. Samples were left to distribute the LifeAct-GFP briefly before imaging. Time-lapse fluorescence imaging was performed on a Nikon Yokogawa CSU-W1 spinning disk confocal using a 20x air objective (Fig. 1E; Extended Data Movie 1, left) or on a Olympus IXplore SpinSR spinning disk confocal using a 20x air objective (Fig. 1F; Extended Data Movie 1, right). A single time point from the imaging is displayed as a z-depth colored intensity projection for Figure 1 E,F.
","reagents":"Phytagel (Sigma-Aldrich P8169)
Old Fashioned Oats (Quaker)
Formaldehyde (Sigma-Aldrich P47608)
1x PBS (adjust to pH 7.8 with KOH)
Rhodamine Phalloidin (Invitrogen R415)
Glass capillaries (World Precision instruments TW100-3)
LifeAct-GFP (250 µM)
","patternDescription":"The macroplasmodial stage of the acellular slime mold Physarum polycephalum is a syncytial reticulated network, with millions of nuclei inhabiting a shared cytoplasm (Fig. 1A; Dove et al., 1986). The cell uses long-range cytoplasmic streaming, with velocities on the order of millimeters per second, to transport bulk material across centimeter-scale intracellular distances (Fig. 1A, B; Kamiya 1959). These substantial cytoplasmic flows are driven by actomyosin-based peristaltic contractions of the cell network, although the molecular and regulatory nature of these contractions remains poorly understood (Fig. 1B; von Olenhusen & Wohlfarth-Bottermann 1979, Kamiya 1981, Alim et al., 2013). Flows transport nutrients and molecular signals across different parts of the polarized cell, from large tube structures at food sources to fan structures at migratory fronts (Fig. 1A; Kramar & Alim 2021). It remains unclear how Physarum compartmentalizes and/or coordinates different actin-based mechanical and structural behaviors across tube and fan structures within a single cytoplasm.
To visualize actin structures across Physarum macroplasmodia (Fig. 1A), we used two parallel approaches: fixed imaging of F-actin via phalloidin staining and live imaging of F-actin via injection of LifeAct-GFP. We grew macroplasmodia on fresh water-agar, then selected and excised large regions of the cell containing both fan and tube structures. These were then either microinjected with LifeAct-GFP or fixed and stained with phalloidin. Below, we characterize in more detail the distinct actin structures found in the tube and fan regions of fixed and live Physarum macroplasmodia.
First, we describe actin structures in feeding tubes. In fixed, phalloidin-labeled tubes, actin was primarily assembled into a membrane-apposed, sheet-like cell cortex (Fig. 1C, yellow and red) and into strand-like circumferential filaments perpendicular to the long axis of the feeding tube (Fig. 1C, blue) as previously characterized (Oettmeier et al., 2018). Live imaging of tubes injected with LifeAct-GFP confirmed the presence of both cortical (Fig. 1E, blue) and circumferential (Fig. 1E, yellow and red) actin structures. In these live specimens, we found that during every contraction/relaxation cycle, the circumferential filaments stretched and shortened while retaining their shape and position (Extended Data Movie 1, left). This finding differs from previous electron microscopy-based characterization of contraction-linked actin structures in Physarum, which emphasized a mechanical function of longitudinal filaments, suggested that filaments fluctuated between linear and mesh-like structures to drive each contraction cycle, and posited a role of dynamic actin filament assembly and disassembly (Nagai et al., 1978, Fleischer & Wohlfarth-Bottermann, 1975). Our fixed and live imaging clarify that circumferential, rather than longitudinal, filaments are the dominant actin structure in tubes and argue against a role of actin network disassembly or restructuring in modulating tube contraction state. This suggests that contraction may instead be regulated by regulated myosin activity, which can generate and relax tension via sliding of adjacent actin filaments without requiring complete network remodeling (Muresan et al., 2022, Quintanilla et al., 2023).
In contrast to the filamentous actin structures in tubes, fixed, phalloidin-labeled fans were instead characterized by an actin cortex with additional patch-like structures (Fig. 1D, light blue, light purple). Additionally, we observed smaller protrusive structures in both fans (Fig 1D, inset) and tubes, reminiscent of filopodia and potentially marking sites of new cell branches. Live imaging of LifeAct-GFP injected fans revealed diffuse cortical actin (Fig. 1F, yellow) as well as dense, fast-traveling actin comets (Fig. 1F, pink and orange; Extended Data Movie 1, right), which were also seen to a lesser extent in tubes (Extended Data Movie 1, left). Together, both live and fixed imaging approaches suggest that actin structures in the fan region of the cell may be specialized to drive migration and endocytosis/exocytosis (Taunton et al., 2000) rather than cytosolic streaming, which has been attributed to the circumferential actin structures present in tubes.
In one living sample, we noticed fan remodeling and retraction away from the area illuminated by microscope light (Extended Data Movie 1, right). During this remodeling event, we observed dense actin comets associated with a transition in actin structure from a sheet-like cortical morphology into filaments (Extended Data Movie 1, right, inset). In other contexts, actin comets are known to play roles in microbe locomotion and intracellular trafficking (Tilney & Portnoy 1989, Giardini et al., 2003), but our observations here implicate them as potential mediators of actin remodeling.
Overall, our work unveils and clarifies the diverse mechanostructural roles of the actin cytoskeleton in the Physarum Polycephalum macroplasmodium. The whole organism contains cortical actin, which hugs the invaginated cell membrane and likely serves a structural role, as well as sparse peripheral filopodial protrusions, which have not yet been described but may serve to nucleate new cell branching events. While tubes are characterized by circumferential actin fibrils that constrict to drive cytoplasmic streaming, fans instead display a patch-like organization and dense actin comets, which may be associated with dynamic network remodeling, migration, and secretion. We find that actin-based structures do not significantly change or remodel across a single contraction cycle, but can convert between cortical, fibrillar, and comet-like morphologies on the scale of tens of minutes. Future work probing the functions of these newly described cytoskeletal elements and uncovering the regulatory pathways that organize such distinct structures within a single cell will prove informative.
","references":[{"reference":"Kamiya N. 1981. Physical and Chemical Basis of Cytoplasmic Streaming. Annual Review of Plant Physiology. 32: 205.","pubmedId":"","doi":"10.1146/annurev.pp.32.060181.001225"},{"reference":"Quintanilla MA, Hammer JA, Beach JR. 2023. Non-muscle myosin 2 at a glance. Journal of Cell Science. 136: jcs260890.","pubmedId":"","doi":"10.1242/jcs.260890"},{"reference":"Muresan CG, Sun ZG, Yadav V, Tabatabai AP, Lanier L, Kim JH, Kim T, Murrell MP. 2022. F-actin architecture determines constraints on myosin thick filament motion. Nature Communications. 13: 7008.","pubmedId":"","doi":"10.1038/s41467-022-34715-6"},{"reference":"Alim K, Amselem G, Peaudecerf F, Brenner MP, Pringle A. 2013. Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual. Proceedings of the National Academy of Sciences. 110: 13306.","pubmedId":"","doi":"10.1073/pnas.1305049110"},{"reference":"Kamiya N. 1959. Protoplasmic Streaming.","pubmedId":"","doi":"10.1007/978-3-7091-5750-3"},{"reference":". 1986. The Molecular Biology of Physarum polycephalum.","pubmedId":"","doi":"10.1007/978-1-4613-2203-0"},{"reference":"Kramar M, Alim K. 2021. Encoding memory in tube diameter hierarchy of living flow network. Proceedings of the National Academy of Sciences. 118: e2007815118.","pubmedId":"","doi":"10.1073/pnas.2007815118"},{"reference":"Nagai R, Yoshimoto Y, Kamiya N. 1978. Cyclic production of tension force in the plasmodial strand of Physarum polycephalum and its relation to microfilament morphology. Journal of Cell Science. 33: 205.","pubmedId":"","doi":"10.1242/jcs.33.1.205"},{"reference":"Fleischer M, Wohlfarth Bottermann KE. 1975. Correlation between tension force generation, fibrillogenesis and ultrastructure of cytoplasmic actomyosin during isometric and isotonic contractions of protoplasmic strands. Cytobiologie. 10: 339.","pubmedId":"","doi":""},{"reference":"Giardini PA, Fletcher DA, Theriot JA. 2003. Compression forces generated by actin comet tails on lipid vesicles. Proceedings of the National Academy of Sciences. 100: 6493.","pubmedId":"","doi":"10.1073/pnas.1031670100"},{"reference":"Tilney LG, Portnoy DA. 1989. Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes.. The Journal of cell biology. 109: 1597.","pubmedId":"","doi":"10.1083/jcb.109.4.1597"},{"reference":"Oettmeier C, Lee J, Dobereiner HG. 2018. Form follows function: ultrastructure of different morphotypes of Physarum polycephalum. Journal of Physics D: Applied Physics. 51: 134006.","pubmedId":"","doi":"10.1088/1361-6463/aab147"},{"reference":"Von Olenhusen KG, Wohlfarth Bottermann KE. 1979. Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell and Tissue Research. 196: 455.","pubmedId":"","doi":"10.1007/BF00234740"},{"reference":"Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA. 2000. Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp✪. The Journal of Cell Biology. 148: 519.","pubmedId":"","doi":"10.1083/jcb.148.3.519"}],"title":"Distinct actin architectures across a giant syncytial cell
","reviews":[{"reviewer":{"displayName":"Christine Field"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"24e5560d-c096-49f2-b31b-7faf494e92eb","decision":"edit","abstract":"The syncytial slime mold Physarum polycephalum exhibits cytosolic streaming, cell migration, and cell network remodeling within a single cell. It is unclear how cytoskeletal elements are spatiotemporally organized to mediate these processes. Here, we combine live and fixed imaging of Physarum F-actin to uncover organizational and dynamic signatures of the actin cytoskeleton across cell structures. We find circumferential actin filaments constrict and relax to drive peristaltic contractions in feeding tubes, while migratory fans are characterized by actin patches and comets. Our work presents undescribed actin structures in Physarum and demonstrates that different actin-based behaviors can coexist within a contiguous cytoplasm.
","acknowledgements":"This work was performed at the 2023 Physiology course at the Marine Biological Laboratory (MBL), Woods Hole, directed by Dr. Nicole King and Dr. Daniel Fletcher, and in the lab of Dr. Amy Gladfelter at Duke University. We thank NIH and MBL for funding and supporting the course that made the work possible, as well as the post-course research. The course was funded by NSF grant #2014108 and Burroughs Wellcome Fund Award # 1282312, We thank Nikon and Olympus for contributing systems to the MBL Physiology course. We thank Rui Jiang and Isaac Breinyn, students of the 2023 MBL Physiology course, for capturing additional movies of Physarum polycephalum that are not included in this manuscript. We also thank the 2023 MBL Physiology course for funding the post-course research of A.C. and Z.G. We thank Dr. Christine Field and Dr. Tim Mitchison for providing LifeAct-GFP protein for injections.
","authors":[{"affiliations":["Columbia University","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Biomedical Engineering","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"adc2174@columbia.edu","firstName":"Andrew D.","lastName":"Countryman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","writing_originalDraft","writing_reviewEditing","visualization"],"email":"zmg2@duke.edu","firstName":"Zachary M.","lastName":"Geisterfer","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States"],"departments":["Department of Cell Biology"],"credit":["investigation","methodology","visualization"],"email":"christine.cao@duke.edu","firstName":"R. Christine","lastName":"Cao","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["supervision","conceptualization","writing_reviewEditing"],"email":"amy.gladfelter@duke.edu","firstName":"Amy S.","lastName":"Gladfelter","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Description: Time-lapse movies of Physarum actin dynamics in tubes (left) and fans (right), as visualized with LifeAct-GFP. The scale bar and time stamps are shared across the two movies.
","doi":null,"resourceType":"Audiovisual","name":"supplementary movie.mp4","url":"https://portal.micropublication.org/uploads/797cd009abcaee7acdd12741de1b62c2.mp4"}],"funding":"The Physiology course was funded by NSF grant # 2014108 and the Burroughs Wellcome Fund Award # 1282312. Z.G is supported by NIH grant # 1F32GM151858-01.
","image":{"url":"https://portal.micropublication.org/uploads/8637ea37f4ad7ce1963a1ee0ede922a5.png"},"imageCaption":"(A) Brightfield image of the macroplasmodium of Physarum polycephalum, with the feeding tubes (blue box) and the migratory fan (red box) marked. Scale bar = 1 mm. (B) Brightfield images with inverted LUT showing the expansion of a feeding tube during peristaltic contractions. Scale bar = 50 µm. (C) Fixed, phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (D) Fixed, phalloidin-labeled actin structures in a migratory fan. Color coding indicates z-depth (see color scale bar). (E) Actin assemblies visualized through LifeAct-GFP in a live feeding tube. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (F) Actin structures, labeled with LifeAct-GFP, in a live feeding fan. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (G-I) Image of a feeding tube costained with phalloidin (magenta) and Hoechst (blue). Scale bar = 15 µm. Inset scale bar = 5 µm.
","imageTitle":"Actin ultrastructure varies across a single Physarum polycephalum macroplasmodium
","methods":"Physarum polycephalum macroplasmodia were grown on 1.7% (wt/vol) low-fluorescence water agar and fed oat flakes every two days. Twenty-four hours before imaging, 1 cm2 agar blocks containing a region of the cell were transferred to a new agar plate and left to develop a mature network in the absence of oat flakes. After starving the sample for 24 hours to reduce autofluorescence, the samples were prepared for live or fixed imaging.
For fixed imaging, sections of the network were prepared by excising an agar block containing structures of interest and gently immersing the entire agar block and specimen into 4% formaldehyde for 10-15 min at RT. After fixation, samples were rinsed three separate times using RT PBS buffer. Each rinse was approximately the volume of the initial formaldehyde immersion and was left for 5 min to allow buffer exchange within the agar block. After rinsing the specimen, samples were immersed in PBS buffer containing either 660 nM Phalloidin-Rhodamine (Fig. 1C,D) or 660 nM Phalloidin-Rhodamine and 500 nM Hoechst 33342 (Fig. 1G–I) and incubated for 10 min at RT. The specimen was rinsed once in PBS as described above, and subsequently all buffer was removed from the imaging well containing the sample. 3D fluorescence microscopy was performed on a Nikon Ti2-E inverted microscope equipped with a Yokogawa CSU-W1 spinning disk confocal. Images were acquired using a 40x Plan Apo silicon immersion objective (NA = 1.25; Fig. 1C,D). Selected images were displayed as a z-depth colored intensity projection for Fig. 1C,D. For fixed imaging of actin and nuclei, fluorescence microscopy was performed on Zeiss LSM 980 confocal. Images were acquired using a 63x Plan-Apochromat oil immersion objective (NA = 1.4; Fig. 1G-I).
For live samples, agar blocks containing regions of interest were transferred to a stereomicroscope (Zeiss Stemi 305) equipped with a FemtoJet 4i microinjector (Eppendorf). Injection needles (World Precision instruments; OD=1.0mm, ID=0.75mm) were pre-loaded with ~3ul of LifeAct-GFP (250 µM) and set to a shallow angle of 5-10° relative to the face of the agar block. The compensation pressure was set to 60 hPa, the injection time was set to 0.5 seconds, and the injection pressure was initially set to 80 hPa. The needle was delicately introduced to a larger tube section of the specimen where the LifeAct-GFP was released. Due to the large intracellular pressure variation within and between Physarum samples, the injection pressure was increased in intervals of 10 hPa until injection resulted in visible release of LifeAct-GFP into the cell. Then, the injection was sustained until reaching a final injection volume of approximately 200 nL. Samples were left to distribute the LifeAct-GFP briefly before imaging. Time-lapse fluorescence imaging was performed on a Nikon Yokogawa CSU-W1 spinning disk confocal using a 20x air objective (Fig. 1E; Extended Data Movie 1, left) or on a Olympus IXplore SpinSR spinning disk confocal using a 20x air objective (Fig. 1F; Extended Data Movie 1, right). A single time point from the imaging is displayed as a z-depth colored intensity projection for Figure 1 E,F.
","reagents":"Phytagel (Sigma-Aldrich P8169)
Old Fashioned Oats (Quaker)
Formaldehyde (Sigma-Aldrich P47608)
1x PBS (adjust to pH 7.8 with KOH)
Rhodamine Phalloidin (Invitrogen R415)
Hoechst 33342 (ThermoFisher H1399)
Glass capillaries (World Precision instruments TW100-3)
LifeAct-GFP (250 µM)
","patternDescription":"The macroplasmodial stage of the acellular slime mold Physarum polycephalum is a syncytial, reticulated network, with millions of nuclei inhabiting a shared cytoplasm (Fig. 1; Dove et al., 1986). The cell uses long-range cytoplasmic streaming, with velocities on the order of millimeters per second, to transport bulk material across centimeter-scale intracellular distances (Fig. 1A,B; Kamiya 1961). These substantial cytoplasmic flows are driven by actomyosin-based peristaltic contractions of the cell network, although the molecular and regulatory nature of these contractions remains poorly understood (Fig. 1B; von Olenhusen & Wohlfarth-Bottermann 1979, Kamiya 1981, Alim et al., 2013). Flows transport nutrients and molecular signals across different parts of the polarized cell, from large tube structures at food sources to fan structures at migratory fronts (Fig. 1A; Kramar & Alim 2021). It remains unclear how Physarum compartmentalizes and/or coordinates different actin-based mechanical and structural behaviors across tube and fan structures within a single cytoplasm.
To visualize actin structures across Physarum macroplasmodia, we used two parallel approaches: fixed imaging of F-actin via phalloidin staining and live imaging of F-actin via injection of LifeAct-GFP. We grew macroplasmodia on fresh water-agar, then selected and excised large regions of the cell containing both fan and tube structures. These were then either microinjected with LifeAct- GFP or fixed and stained with phalloidin. Below, we characterize in more detail the distinct actin structures found in the tube and fan regions of fixed and live Physarum macroplasmodia.
First, we describe actin structures in feeding tubes. In fixed, phalloidin-labeled tubes, actin was primarily assembled into a sheet-like cell cortex and into strand-like circumferential filaments perpendicular to the long axis of the feeding tube, as previously characterized (Fig. 1C, teal; Oettmeier et al., 2018). Live imaging of tubes injected with LifeAct-GFP confirmed the presence of both sheet-like cortical (Fig. 1E, teal) and filamentous circumferential (Fig. 1E, magenta) actin structures. In these live specimens, we found that during every contraction/relaxation cycle, the circumferential filaments stretched and shortened while retaining their shape and position (Extended Data Movie 1, left). This finding differs from previous electron microscopy-based characterization of contraction-linked actin structures in Physarum, which emphasized a mechanical function of longitudinal filaments, suggested that filaments fluctuated between linear and mesh-like structures to drive each contraction cycle, and posited a role of dynamic actin filament assembly and disassembly (Nagai et al., 1978, Fleischer & Wohlfarth-Bottermann, 1975). Our fixed and live imaging clarify that circumferential, rather than longitudinal, filaments are the dominant actin structure in tubes and argue against a role of actin network disassembly or restructuring in modulating tube contraction state. This suggests that contraction may instead be regulated by regulated myosin activity, which can generate and relax tension via sliding of adjacent actin filaments without requiring complete network remodeling (Muresan et al., 2022, Quintanilla et al., 2023).
In contrast to the filamentous actin structures in tubes, fixed, phalloidin-labeled fans were instead characterized by an actin cortex with additional patch-like structures (Fig. 1D). Additionally, we observed smaller protrusive structures in both fans (Fig 1D, inset) and tubes, reminiscent of filopodia and potentially marking sites of new cell branches. Live imaging of fans injected with LifeAct-GFP revealed diffuse cortical actin as well as dense, fast-traveling actin comets (Fig. 1F; Extended Data Movie 1, right), which were also seen to a lesser extent in tubes (Extended Data Movie 1, left). In one living sample, we noticed fan remodeling and retraction away from the area illuminated by microscope light (Extended Data Movie 1, right). During this remodeling event, we observed dense actin comets associated with a transition in actin structure from a sheet-like cortical morphology into filaments (Extended Data Movie 1, right, inset). In other contexts, actin comets are known to play roles in microbe locomotion and intracellular trafficking (Tilney & Portnoy 1989, Giardini et al., 2003), but our observations here implicate them as potential mediators of actin remodeling. Together, both live and fixed imaging approaches suggest that actin structures in the fan region of the cell may be specialized to drive migration and endocytosis/exocytosis (Taunton et al., 2000) rather than cytosolic streaming, which has been attributed to the circumferential actin structures present in tubes.
To understand how nuclei were positioned with respect to the actin cytoskeleton in this syncytial cell, we co-stained fixed specimens with phalloidin and Hoechst. We found that nuclei were distributed throughout the cell, occupying phalloidin-depleted regions within an otherwise actin-rich cytoplasm (Fig. 1G–I). While it is possible that actin may play a role in nuclear positioning, we did not observe any obvious, stereotyped actin structures associated with individual nuclei. Further studies will be necessary to explore the functional importance of actin in positioning organelles under substantial cytoplasmic flow.
Overall, our work unveils and clarifies the diverse mechanostructural roles of the actin cytoskeleton in the Physarum Polycephalum macroplasmodium. The whole organism contains cortical actin, which hugs the invaginated cell membrane and likely serves to position organelles and provide structural support to the cell, as well as sparse peripheral filopodial protrusions, which have not yet been described but may serve to nucleate new cell branching events. While tubes are characterized by circumferential actin filaments that constrict to drive cytoplasmic streaming, fans instead display a patch-like organization and dense actin comets, which may be associated with dynamic network remodeling, migration, and secretion. We find that actin-based structures do not significantly change or remodel across a single contraction cycle, but can convert between cortical, filamentous, and comet-like morphologies on the scale of tens of minutes. Future work probing the functions of these newly described cytoskeletal elements and uncovering the regulatory pathways that organize such distinct structures within a single cell will prove informative.
","references":[{"reference":"Dove, W. F., Dee, J., Hatano, S., Haugli, F. B., & Wohlfarth-Bottermann, K.-E. (Eds.). (1986). The Molecular Biology of Physarum polycephalum. Springer New York.
","pubmedId":"","doi":"10.1007/978-1-4613-2203-0"},{"reference":"Alim K, Amselem G, Peaudecerf F, Brenner MP, Pringle A. (2013). Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual. Proceedings of the National Academy of Sciences, 110(33), 13306–13311.
","pubmedId":"","doi":"10.1073/pnas.1305049110"},{"reference":"Fleischer M, Wohlfarth Bottermann KE. (1975). Correlation between tension force generation, fibrillogenesis and ultrastructure of cytoplasmic actomyosin during isometric and isotonic contractions of protoplasmic strands. Cytobiologie, 10(3), 339–365.
","pubmedId":"","doi":""},{"reference":"Giardini PA, Fletcher DA, Theriot JA. (2003). Compression forces generated by actin comet tails on lipid vesicles. Proceedings of the National Academy of Sciences, 100(11), 6493–6498.
","pubmedId":"","doi":"10.1073/pnas.1031670100"},{"reference":"Kamiya N. (1981). Physical and Chemical Basis of Cytoplasmic Streaming. Annual Review of Plant Physiology, , 32(1), 205–236.
","pubmedId":"","doi":"10.1146/annurev.pp.32.060181.001225"},{"reference":"Kamiya, N. (1961). Protoplasmic streaming. Protoplasma, (53), 600-614.
","pubmedId":"","doi":"10.1007/BF01248235"},{"reference":"Kramar M, Alim K. (2021). Encoding memory in tube diameter hierarchy of living flow network. Proceedings of the National Academy of Sciences, 118(10), e2007815118.
","pubmedId":"","doi":"10.1073/pnas.2007815118"},{"reference":"Muresan CG, Sun ZG, Yadav V, Tabatabai AP, Lanier L, Kim JH, Kim T, Murrell MP. (2022). F-actin architecture determines constraints on myosin thick filament motion. Nature Communications, 13(1), 7008.
","pubmedId":"","doi":"10.1038/s41467-022-34715-6"},{"reference":"Nagai R, Yoshimoto Y, Kamiya N. (1978). Cyclic production of tension force in the plasmodial strand of Physarum polycephalum and its relation to microfilament morphology. Journal of Cell Science, 33(1), 205–225.
","pubmedId":"","doi":"10.1242/jcs.33.1.205"},{"reference":"Oettmeier C, Lee J, Dobereiner HG. (2018). Form follows function: ultrastructure of different morphotypes of Physarum polycephalum. Journal of Physics D: Applied Physics, 51(13), 134006.
","pubmedId":"","doi":"10.1088/1361-6463/aab147"},{"reference":"Quintanilla MA, Hammer JA, Beach JR. (2023). Non-muscle myosin 2 at a glance. Journal of Cell Science, 136(5), jcs260890.
","pubmedId":"","doi":"10.1242/jcs.260890"},{"reference":"Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA. (2000). Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp. The Journal of Cell Biology, 148(3), 519-530.
","pubmedId":"","doi":"10.1083/jcb.148.3.519"},{"reference":"Tilney LG, Portnoy DA. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes.. The Journal of cell biology, 109(4), 1597–1608.
","pubmedId":"","doi":"10.1083/jcb.109.4.1597"},{"reference":"Von Olenhusen KG, Wohlfarth Bottermann KE. (1979). Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell and Tissue Research, 196(3), 455–470.
","pubmedId":"","doi":"10.1007/BF00234740"}],"title":"Distinct actin architectures across a giant syncytial cell
","reviews":[{"reviewer":{"displayName":"Christine Field"},"openAcknowledgement":false,"status":{"submitted":true}}],"curatorReviews":[]},{"id":"170a7be1-80f5-495c-b161-4ca8b89cdd1a","decision":"accept","abstract":"The syncytial slime mold Physarum polycephalum exhibits cytosolic streaming, cell migration, and cell network remodeling within a single cell. It is unclear how cytoskeletal elements are spatiotemporally organized to mediate these processes. Here, we combine live and fixed imaging of Physarum F-actin to uncover organizational and dynamic signatures of the actin cytoskeleton across cell structures. We find circumferential actin filaments constrict and relax to drive peristaltic contractions in feeding tubes, while migratory fans are characterized by actin patches and comets. Our work presents undescribed actin structures in Physarum and demonstrates that different actin-based behaviors can coexist within a contiguous cytoplasm.
","acknowledgements":"This work was performed at the 2023 Physiology course at the Marine Biological Laboratory (MBL), Woods Hole, directed by Dr. Nicole King and Dr. Daniel Fletcher, and in the lab of Dr. Amy Gladfelter at Duke University. We thank NIH and MBL for funding and supporting the course that made the work possible, as well as the post-course research. The course was funded by NSF grant #2014108 and Burroughs Wellcome Fund Award # 1282312, We thank Nikon and Olympus for contributing systems to the MBL Physiology course. We thank Rui Jiang and Isaac Breinyn, students of the 2023 MBL Physiology course, for capturing additional movies of Physarum polycephalum that are not included in this manuscript. We also thank the 2023 MBL Physiology course for funding the post-course research of A.C. and Z.G. We thank Dr. Christine Field and Dr. Tim Mitchison for providing LifeAct-GFP protein for injections.
","authors":[{"affiliations":["Columbia University, New York, NY, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Biomedical Engineering","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"adc2174@columbia.edu","firstName":"Andrew D.","lastName":"Countryman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","writing_originalDraft","writing_reviewEditing","visualization"],"email":"zmg2@duke.edu","firstName":"Zachary M.","lastName":"Geisterfer","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States"],"departments":["Department of Cell Biology"],"credit":["investigation","methodology","visualization"],"email":"christine.cao@duke.edu","firstName":"R. Christine","lastName":"Cao","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["supervision","conceptualization","writing_reviewEditing"],"email":"amy.gladfelter@duke.edu","firstName":"Amy S.","lastName":"Gladfelter","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Description: Time-lapse movies of Physarum actin dynamics in tubes (left) and fans (right), as visualized with LifeAct-GFP. The scale bar and time stamps are shared across the two movies.
","doi":"10.22002/8axsk-46644","resourceType":"Audiovisual","name":"supplementary movie.mp4","url":"https://portal.micropublication.org/uploads/797cd009abcaee7acdd12741de1b62c2.mp4"}],"funding":"The Physiology course was funded by NSF grant # 2014108 and the Burroughs Wellcome Fund Award # 1282312. Z.G is supported by NIH grant # 1F32GM151858-01.
","image":{"url":"https://portal.micropublication.org/uploads/8637ea37f4ad7ce1963a1ee0ede922a5.png"},"imageCaption":"(A) Brightfield image of the macroplasmodium of Physarum polycephalum, with the feeding tubes (blue box) and the migratory fan (red box) marked. Scale bar = 1 mm. (B) Brightfield images with inverted LUT showing the expansion of a feeding tube during peristaltic contractions. Scale bar = 50 µm. (C) Fixed, phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (D) Fixed, phalloidin-labeled actin structures in a migratory fan. Color coding indicates z-depth (see color scale bar). (E) Actin assemblies visualized through LifeAct-GFP in a live feeding tube. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (F) Actin structures, labeled with LifeAct-GFP, in a live feeding fan. 3D images taken from Extended data video 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (G-I) Image of a feeding tube costained with phalloidin (magenta) and Hoechst (blue). Scale bar = 15 µm. Inset scale bar = 5 µm.
","imageTitle":"Actin ultrastructure varies across a single Physarum polycephalum macroplasmodium
","methods":"Physarum polycephalum macroplasmodia were grown on 1.7% (wt/vol) low-fluorescence water agar and fed oat flakes every two days. Twenty-four hours before imaging, 1 cm2 agar blocks containing a region of the cell were transferred to a new agar plate and left to develop a mature network in the absence of oat flakes. After starving the sample for 24 hours to reduce autofluorescence, the samples were prepared for live or fixed imaging.
For fixed imaging, sections of the network were prepared by excising an agar block containing structures of interest and gently immersing the entire agar block and specimen into 4% formaldehyde for 10-15 min at RT. After fixation, samples were rinsed three separate times using RT PBS buffer. Each rinse was approximately the volume of the initial formaldehyde immersion and was left for 5 min to allow buffer exchange within the agar block. After rinsing the specimen, samples were immersed in PBS buffer containing either 660 nM Phalloidin-Rhodamine (Fig. 1C,D) or 660 nM Phalloidin-Rhodamine and 500 nM Hoechst 33342 (Fig. 1G–I) and incubated for 10 min at RT. The specimen was rinsed once in PBS as described above, and subsequently all buffer was removed from the imaging well containing the sample. 3D fluorescence microscopy was performed on a Nikon Ti2-E inverted microscope equipped with a Yokogawa CSU-W1 spinning disk confocal. Images were acquired using a 40x Plan Apo silicon immersion objective (NA = 1.25; Fig. 1C,D). Selected images were displayed as a z-depth colored intensity projection for Fig. 1C,D. For fixed imaging of actin and nuclei, fluorescence microscopy was performed on Zeiss LSM 980 confocal. Images were acquired using a 63x Plan-Apochromat oil immersion objective (NA = 1.4; Fig. 1G-I).
For live samples, agar blocks containing regions of interest were transferred to a stereomicroscope (Zeiss Stemi 305) equipped with a FemtoJet 4i microinjector (Eppendorf). Injection needles (World Precision instruments; OD=1.0mm, ID=0.75mm) were pre-loaded with ~3ul of LifeAct-GFP (250 µM) and set to a shallow angle of 5-10° relative to the face of the agar block. The compensation pressure was set to 60 hPa, the injection time was set to 0.5 seconds, and the injection pressure was initially set to 80 hPa. The needle was delicately introduced to a larger tube section of the specimen where the LifeAct-GFP was released. Due to the large intracellular pressure variation within and between Physarum samples, the injection pressure was increased in intervals of 10 hPa until injection resulted in visible release of LifeAct-GFP into the cell. Then, the injection was sustained until reaching a final injection volume of approximately 200 nL. Samples were left to distribute the LifeAct-GFP briefly before imaging. Time-lapse fluorescence imaging was performed on a Nikon Yokogawa CSU-W1 spinning disk confocal using a 20x air objective (Fig. 1E; Extended Data Movie 1, left) or on a Olympus IXplore SpinSR spinning disk confocal using a 20x air objective (Fig. 1F; Extended Data Movie 1, right). A single time point from the imaging is displayed as a z-depth colored intensity projection for Figure 1 E,F.
","reagents":"Phytagel (Sigma-Aldrich P8169)
Old Fashioned Oats (Quaker)
Formaldehyde (Sigma-Aldrich P47608)
1x PBS (adjust to pH 7.8 with KOH)
Rhodamine Phalloidin (Invitrogen R415)
Hoechst 33342 (ThermoFisher H1399)
Glass capillaries (World Precision instruments TW100-3)
LifeAct-GFP (250 µM)
","patternDescription":"The macroplasmodial stage of the acellular slime mold Physarum polycephalum is a syncytial, reticulated network, with millions of nuclei inhabiting a shared cytoplasm (Fig. 1; Dove et al., 1986). The cell uses long-range cytoplasmic streaming, with velocities on the order of millimeters per second, to transport bulk material across centimeter-scale intracellular distances (Fig. 1A,B; Kamiya 1961). These substantial cytoplasmic flows are driven by actomyosin-based peristaltic contractions of the cell network, although the molecular and regulatory nature of these contractions remains poorly understood (Fig. 1B; von Olenhusen & Wohlfarth-Bottermann 1979, Kamiya 1981, Alim et al., 2013). Flows transport nutrients and molecular signals across different parts of the polarized cell, from large tube structures at food sources to fan structures at migratory fronts (Fig. 1A; Kramar & Alim 2021). It remains unclear how Physarum compartmentalizes and/or coordinates different actin-based mechanical and structural behaviors across tube and fan structures within a single cytoplasm.
To visualize actin structures across Physarum macroplasmodia, we used two parallel approaches: fixed imaging of F-actin via phalloidin staining and live imaging of F-actin via injection of LifeAct-GFP. We grew macroplasmodia on fresh water-agar, then selected and excised large regions of the cell containing both fan and tube structures. These were then either microinjected with LifeAct- GFP or fixed and stained with phalloidin. Below, we characterize in more detail the distinct actin structures found in the tube and fan regions of fixed and live Physarum macroplasmodia.
First, we describe actin structures in feeding tubes. In fixed, phalloidin-labeled tubes, actin was primarily assembled into a sheet-like cell cortex and into strand-like circumferential filaments perpendicular to the long axis of the feeding tube, as previously characterized (Fig. 1C, teal; Oettmeier et al., 2018). Live imaging of tubes injected with LifeAct-GFP confirmed the presence of both sheet-like cortical (Fig. 1E, teal) and filamentous circumferential (Fig. 1E, magenta) actin structures. In these live specimens, we found that during every contraction/relaxation cycle, the circumferential filaments stretched and shortened while retaining their shape and position (Extended Data Movie 1, left). This finding differs from previous electron microscopy-based characterization of contraction-linked actin structures in Physarum, which emphasized a mechanical function of longitudinal filaments, suggested that filaments fluctuated between linear and mesh-like structures to drive each contraction cycle, and posited a role of dynamic actin filament assembly and disassembly (Nagai et al., 1978, Fleischer & Wohlfarth-Bottermann, 1975). Our fixed and live imaging clarify that circumferential, rather than longitudinal, filaments are the dominant actin structure in tubes and argue against a role of actin network disassembly or restructuring in modulating tube contraction state. This suggests that contraction may instead be regulated by regulated myosin activity, which can generate and relax tension via sliding of adjacent actin filaments without requiring complete network remodeling (Muresan et al., 2022, Quintanilla et al., 2023).
In contrast to the filamentous actin structures in tubes, fixed, phalloidin-labeled fans were instead characterized by an actin cortex with additional patch-like structures (Fig. 1D). Additionally, we observed smaller protrusive structures in both fans (Fig 1D, inset) and tubes, reminiscent of filopodia and potentially marking sites of new cell branches. Live imaging of fans injected with LifeAct-GFP revealed diffuse cortical actin as well as dense, fast-traveling actin comets (Fig. 1F; Extended Data Movie 1, right), which were also seen to a lesser extent in tubes (Extended Data Movie 1, left). In one living sample, we noticed fan remodeling and retraction away from the area illuminated by microscope light (Extended Data Movie 1, right). During this remodeling event, we observed dense actin comets associated with a transition in actin structure from a sheet-like cortical morphology into filaments (Extended Data Movie 1, right, inset). In other contexts, actin comets are known to play roles in microbe locomotion and intracellular trafficking (Tilney & Portnoy 1989, Giardini et al., 2003), but our observations here implicate them as potential mediators of actin remodeling. Together, both live and fixed imaging approaches suggest that actin structures in the fan region of the cell may be specialized to drive migration and endocytosis/exocytosis (Taunton et al., 2000) rather than cytosolic streaming, which has been attributed to the circumferential actin structures present in tubes.
To understand how nuclei were positioned with respect to the actin cytoskeleton in this syncytial cell, we co-stained fixed specimens with phalloidin and Hoechst. We found that nuclei were distributed throughout the cell, occupying phalloidin-depleted regions within an otherwise actin-rich cytoplasm (Fig. 1G–I). While it is possible that actin may play a role in nuclear positioning, we did not observe any obvious, stereotyped actin structures associated with individual nuclei. Further studies will be necessary to explore the functional importance of actin in positioning organelles under substantial cytoplasmic flow.
Overall, our work unveils and clarifies the diverse mechanostructural roles of the actin cytoskeleton in the Physarum Polycephalum macroplasmodium. The whole organism contains cortical actin, which hugs the invaginated cell membrane and likely serves to position organelles and provide structural support to the cell, as well as sparse peripheral filopodial protrusions, which have not yet been described but may serve to nucleate new cell branching events. While tubes are characterized by circumferential actin filaments that constrict to drive cytoplasmic streaming, fans instead display a patch-like organization and dense actin comets, which may be associated with dynamic network remodeling, migration, and secretion. We find that actin-based structures do not significantly change or remodel across a single contraction cycle, but can convert between cortical, filamentous, and comet-like morphologies on the scale of tens of minutes. Future work probing the functions of these newly described cytoskeletal elements and uncovering the regulatory pathways that organize such distinct structures within a single cell will prove informative.
","references":[{"reference":"Dove, W. F., Dee, J., Hatano, S., Haugli, F. B., & Wohlfarth-Bottermann, K.-E. (Eds.). (1986). The Molecular Biology of Physarum polycephalum. Springer New York.
","pubmedId":"","doi":"10.1007/978-1-4613-2203-0"},{"reference":"Alim K, Amselem G, Peaudecerf F, Brenner MP, Pringle A. (2013). Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual. Proceedings of the National Academy of Sciences, 110(33), 13306–13311.
","pubmedId":"","doi":"10.1073/pnas.1305049110"},{"reference":"Fleischer M, Wohlfarth Bottermann KE. (1975). Correlation between tension force generation, fibrillogenesis and ultrastructure of cytoplasmic actomyosin during isometric and isotonic contractions of protoplasmic strands. Cytobiologie, 10(3), 339–365.
","pubmedId":"","doi":""},{"reference":"Giardini PA, Fletcher DA, Theriot JA. (2003). Compression forces generated by actin comet tails on lipid vesicles. Proceedings of the National Academy of Sciences, 100(11), 6493–6498.
","pubmedId":"","doi":"10.1073/pnas.1031670100"},{"reference":"Kamiya N. (1981). Physical and Chemical Basis of Cytoplasmic Streaming. Annual Review of Plant Physiology, , 32(1), 205–236.
","pubmedId":"","doi":"10.1146/annurev.pp.32.060181.001225"},{"reference":"Kamiya, N. (1961). Protoplasmic streaming. Protoplasma, (53), 600-614.
","pubmedId":"","doi":"10.1007/BF01248235"},{"reference":"Kramar M, Alim K. (2021). Encoding memory in tube diameter hierarchy of living flow network. Proceedings of the National Academy of Sciences, 118(10), e2007815118.
","pubmedId":"","doi":"10.1073/pnas.2007815118"},{"reference":"Muresan CG, Sun ZG, Yadav V, Tabatabai AP, Lanier L, Kim JH, Kim T, Murrell MP. (2022). F-actin architecture determines constraints on myosin thick filament motion. Nature Communications, 13(1), 7008.
","pubmedId":"","doi":"10.1038/s41467-022-34715-6"},{"reference":"Nagai R, Yoshimoto Y, Kamiya N. (1978). Cyclic production of tension force in the plasmodial strand of Physarum polycephalum and its relation to microfilament morphology. Journal of Cell Science, 33(1), 205–225.
","pubmedId":"","doi":"10.1242/jcs.33.1.205"},{"reference":"Oettmeier C, Lee J, Dobereiner HG. (2018). Form follows function: ultrastructure of different morphotypes of Physarum polycephalum. Journal of Physics D: Applied Physics, 51(13), 134006.
","pubmedId":"","doi":"10.1088/1361-6463/aab147"},{"reference":"Quintanilla MA, Hammer JA, Beach JR. (2023). Non-muscle myosin 2 at a glance. Journal of Cell Science, 136(5), jcs260890.
","pubmedId":"","doi":"10.1242/jcs.260890"},{"reference":"Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA. (2000). Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp. The Journal of Cell Biology, 148(3), 519-530.
","pubmedId":"","doi":"10.1083/jcb.148.3.519"},{"reference":"Tilney LG, Portnoy DA. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes.. The Journal of cell biology, 109(4), 1597–1608.
","pubmedId":"","doi":"10.1083/jcb.109.4.1597"},{"reference":"Von Olenhusen KG, Wohlfarth Bottermann KE. (1979). Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell and Tissue Research, 196(3), 455–470.
","pubmedId":"","doi":"10.1007/BF00234740"}],"title":"Distinct actin architectures across a giant syncytial cell
","reviews":[],"curatorReviews":[]},{"id":"2d3626e9-8ad4-4fe1-a824-27380f5be56d","decision":"edit","abstract":"The syncytial slime mold Physarum polycephalum exhibits cytosolic streaming, cell migration, and cell network remodeling within a single cell. It is unclear how cytoskeletal elements are spatiotemporally organized to mediate these processes. Here, we combine live and fixed imaging of Physarum F-actin to uncover organizational and dynamic signatures of the actin cytoskeleton across cell structures. We find circumferential actin filaments constrict and relax to drive peristaltic contractions in feeding tubes, while migratory fans are characterized by actin patches and comets. Our work presents undescribed actin structures in Physarum and demonstrates that different actin-based behaviors coexist within a contiguous cytoplasm.
","acknowledgements":"This work was performed at the 2023 Physiology course at the Marine Biological Laboratory (MBL), Woods Hole, directed by Dr. Nicole King and Dr. Daniel Fletcher, and in the lab of Dr. Amy Gladfelter at Duke University. We thank NIH and MBL for funding and supporting the course that made the work possible, as well as the post-course research. The course was funded by NSF grant #2014108 and Burroughs Wellcome Fund Award # 1282312, We thank Nikon and Olympus for contributing systems to the MBL Physiology course. We thank Rui Jiang and Isaac Breinyn, students of the 2023 MBL Physiology course, for capturing additional movies of Physarum polycephalum that are not included in this manuscript. We also thank the 2023 MBL Physiology course for funding the post-course research of A.C. and Z.G. We thank Dr. Christine Field and Dr. Tim Mitchison for providing LifeAct-GFP protein for injections.
","authors":[{"affiliations":["Columbia University, New York, NY, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Biomedical Engineering","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"adc2174@columbia.edu","firstName":"Andrew D.","lastName":"Countryman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","writing_originalDraft","writing_reviewEditing","visualization"],"email":"zmg2@duke.edu","firstName":"Zachary M.","lastName":"Geisterfer","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States"],"departments":["Department of Cell Biology"],"credit":["investigation","methodology","visualization"],"email":"christine.cao@duke.edu","firstName":"R. Christine","lastName":"Cao","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["supervision","conceptualization","writing_reviewEditing"],"email":"amy.gladfelter@duke.edu","firstName":"Amy S.","lastName":"Gladfelter","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Time-lapse movies of Physarum actin dynamics in tubes (left) and fans (right), as visualized with LifeAct-GFP. The scale bar and time stamps are shared across the two movies.
","doi":null,"resourceType":"Audiovisual","name":"Extended_Data_Movie_1.mp4","url":"https://portal.micropublication.org/uploads/1a4b4b3281507c1a6d4f8c3fb8565725.mp4"}],"funding":"The Physiology course was funded by NSF grant # 2014108 and the Burroughs Wellcome Fund Award # 1282312. Z.G is supported by NIH grant # 1F32GM151858-01.
","image":{"url":"https://portal.micropublication.org/uploads/f41c89c5b8af9fb2ac1cfc922b35edd4.png"},"imageCaption":"(A) Brightfield image of the macroplasmodium of Physarum polycephalum, with the feeding tubes (blue box) and the migratory fan (red box) marked. Scale bar = 1 mm. (B) Brightfield images with inverted LUT showing the expansion of a feeding tube during peristaltic contractions. Scale bar = 50 µm. (C) Fixed, phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (D) Fixed, phalloidin-labeled actin structures in a migratory fan. Color coding indicates z-depth (see color scale bar). (E) Actin assemblies visualized through LifeAct-GFP in a live feeding tube. 3D images taken from Extended Data Movie 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (F) Actin structures, labeled with LifeAct-GFP, in a live feeding fan. 3D images taken from Extended Data Movie 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (G-I) Image of a feeding tube costained with phalloidin (magenta) and Hoechst (blue). Scale bar = 15 µm. Inset scale bar = 5 µm.
","imageTitle":"Actin ultrastructure varies across a single Physarum polycephalum macroplasmodium
","methods":"Physarum polycephalum macroplasmodia were grown on 1.7% (wt/vol) low-fluorescence water agar and fed oat flakes every two days. Twenty-four hours before imaging, 1 cm2 agar blocks containing a region of the cell were transferred to a new agar plate and left to develop a mature network in the absence of oat flakes. After starving the sample for 24 hours to reduce autofluorescence, the samples were prepared for live or fixed imaging.
For fixed imaging, sections of the network were prepared by excising an agar block containing structures of interest and gently immersing the entire agar block and specimen into 4% formaldehyde for 10-15 min at RT. After fixation, samples were rinsed three separate times using RT PBS buffer. Each rinse was approximately the volume of the initial formaldehyde immersion and was left for 5 min to allow buffer exchange within the agar block. After rinsing the specimen, samples were immersed in PBS buffer containing either 660 nM Phalloidin-Rhodamine (Fig. 1C,D) or 660 nM Phalloidin-Rhodamine and 500 nM Hoechst 33342 (Fig. 1G–I) and incubated for 10 min at RT. The specimen was rinsed once in PBS as described above, and subsequently all buffer was removed from the imaging well containing the sample. 3D fluorescence microscopy was performed on a Nikon Ti2-E inverted microscope equipped with a Yokogawa CSU-W1 spinning disk confocal. Images were acquired using a 40x Plan Apo silicon immersion objective (NA = 1.25; Fig. 1C,D). Selected images were displayed as a z-depth colored intensity projection for Fig. 1C,D. For fixed imaging of actin and nuclei, fluorescence microscopy was performed on Zeiss LSM 980 confocal. Images were acquired using a 63x Plan-Apochromat oil immersion objective (NA = 1.4; Fig. 1G-I).
For live samples, agar blocks containing regions of interest were transferred to a stereomicroscope (Zeiss Stemi 305) equipped with a FemtoJet 4i microinjector (Eppendorf). Injection needles (World Precision instruments; OD=1.0mm, ID=0.75mm) were pre-loaded with ~3 μl of LifeAct-GFP (250 µM) and set to a shallow angle of 5-10° relative to the face of the agar block. The compensation pressure was set to 60 hPa, the injection time was set to 0.5 seconds, and the injection pressure was initially set to 80 hPa. The needle was delicately introduced to a larger tube section of the specimen where the LifeAct-GFP was released. Due to the large intracellular pressure variation within and between Physarum samples, the injection pressure was increased in intervals of 10 hPa until injection resulted in visible release of LifeAct-GFP into the cell. Then, the injection was sustained until reaching a final injection volume of approximately 200 nL. Samples were left to distribute the LifeAct-GFP briefly before imaging. Time-lapse fluorescence imaging was performed on a Nikon Yokogawa CSU-W1 spinning disk confocal using a 20x air objective (Fig. 1E; Extended Data Movie 1, left) or on an Olympus IXplore SpinSR spinning disk confocal using a 20x air objective (Fig. 1F; Extended Data Movie 1, right). A single time point from the imaging is displayed as a z-depth colored intensity projection for Figure 1 E,F.
","reagents":"Phytagel (Sigma-Aldrich P8169)
Old Fashioned Oats (Quaker)
Formaldehyde (Sigma-Aldrich P47608)
1x PBS (adjust to pH 7.8 with KOH)
Rhodamine Phalloidin (Invitrogen R415)
Hoechst 33342 (ThermoFisher H1399)
Glass capillaries (World Precision instruments TW100-3)
LifeAct-GFP (250 µM)
","patternDescription":"The macroplasmodial stage of the acellular slime mold Physarum polycephalum is a syncytial, reticulated network, with millions of nuclei inhabiting a shared cytoplasm (Fig. 1; Dove et al., 1986). The cell uses long-range cytoplasmic streaming, with velocities on the order of millimeters per second, to transport bulk material across centimeter-scale intracellular distances (Fig. 1A,B; Kamiya 1961). These substantial cytoplasmic flows are driven by actomyosin-based peristaltic contractions of the cell network, although the molecular and regulatory nature of these contractions remains poorly understood (Fig. 1B; von Olenhusen & Wohlfarth-Bottermann 1979, Kamiya 1981, Alim et al., 2013). Flows transport nutrients and molecular signals across different parts of the polarized cell, from large tube structures at food sources to fan structures at migratory fronts (Fig. 1A; Kramar & Alim 2021). It remains unclear how Physarum compartmentalizes and/or coordinates different actin-based mechanical and structural behaviors across tube and fan structures within a single cytoplasm.
To visualize actin structures across Physarum macroplasmodia, we used two parallel approaches: fixed imaging of F-actin via phalloidin staining and live imaging of F-actin via injection of LifeAct-GFP. We grew macroplasmodia on fresh water-agar, then selected and excised large regions of the cell containing both fan and tube structures. These were then either microinjected with LifeAct- GFP or fixed and stained with phalloidin. Below, we characterize in more detail the distinct actin structures found in the tube and fan regions of fixed and live Physarum macroplasmodia.
First, we describe actin structures in feeding tubes. In fixed, phalloidin-labeled tubes, actin was primarily assembled into a sheet-like cell cortex and into strand-like circumferential filaments perpendicular to the long axis of the feeding tube, as previously characterized (Fig. 1C, teal; Oettmeier et al., 2018). Live imaging of tubes injected with LifeAct-GFP confirmed the presence of both sheet-like cortical (Fig. 1E, teal) and filamentous circumferential (Fig. 1E, magenta) actin structures. In these live specimens, we found that during every contraction/relaxation cycle, the circumferential filaments stretched and shortened while retaining their shape and position (Extended Data Movie 1, left). This finding differs from previous electron microscopy-based characterization of contraction-linked actin structures in Physarum, which emphasized a mechanical function of longitudinal filaments, suggested that filaments fluctuated between linear and mesh-like structures to drive each contraction cycle, and posited a role of dynamic actin filament assembly and disassembly (Nagai et al., 1978, Fleischer & Wohlfarth-Bottermann, 1975). Our fixed and live imaging clarify that circumferential, rather than longitudinal, filaments are the dominant actin structure in tubes and argue against a role of actin network disassembly or restructuring in modulating tube contraction state. This suggests that contraction may instead be controlled by regulated myosin activity, which can generate and relax tension via sliding of adjacent actin filaments without requiring complete network remodeling (Muresan et al., 2022, Quintanilla et al., 2023).
In contrast to the filamentous actin structures in tubes, fixed, phalloidin-labeled fans were instead characterized by an actin cortex with additional patch-like structures (Fig. 1D). Additionally, we observed smaller protrusive structures in both fans (Fig 1D, inset) and tubes, reminiscent of filopodia and potentially marking sites of new cell branches. Live imaging of fans injected with LifeAct-GFP revealed diffuse cortical actin as well as dense, fast-traveling actin comets (Fig. 1F; Extended Data Movie 1, right), which were also seen to a lesser extent in tubes (Extended Data Movie 1, left). In one living sample, we noticed fan remodeling and retraction away from the area illuminated by microscope light (Extended Data Movie 1, right). During this remodeling event, we observed dense actin comets associated with a transition in actin structure from a sheet-like cortical morphology into filaments (Extended Data Movie 1, right, inset). In other contexts, actin comets are known to play roles in microbe locomotion and intracellular trafficking (Tilney & Portnoy 1989, Giardini et al., 2003), but our observations here implicate them as potential mediators of actin remodeling. Together, both live and fixed imaging approaches suggest that actin structures in the fan region of the cell may be specialized to drive migration and endocytosis/exocytosis (Taunton et al., 2000) rather than cytosolic streaming, which has been attributed to the circumferential actin structures present in tubes.
To understand how nuclei were positioned with respect to the actin cytoskeleton in this syncytial cell, we co-stained fixed specimens with phalloidin and Hoechst. We found that nuclei were distributed throughout the cell, occupying phalloidin-depleted regions within an otherwise actin-rich cytoplasm (Fig. 1G–I). While it is possible that actin may play a role in nuclear positioning, we did not observe any obvious, stereotyped actin structures associated with individual nuclei. Further studies will be necessary to explore the functional importance of actin in positioning organelles under substantial cytoplasmic flow.
Overall, our work unveils and clarifies the diverse mechanostructural roles of the actin cytoskeleton in the Physarum polycephalum macroplasmodium. The whole organism contains cortical actin, which hugs the invaginated cell membrane and likely serves to position organelles and provide structural support to the cell, as well as sparse peripheral filopodial protrusions, which have not yet been described but may serve to nucleate new cell branching events. While tubes are characterized by circumferential actin filaments that constrict to drive cytoplasmic streaming, fans instead display a patch-like organization and dense actin comets, which may be associated with dynamic network remodeling, migration, and secretion. We find that actin-based structures do not significantly change or remodel across a single contraction cycle, but can convert between cortical, filamentous, and comet-like morphologies on the scale of tens of minutes. Future work probing the functions of these newly described cytoskeletal elements and uncovering the regulatory pathways that organize such distinct structures within a single cell will prove informative.
","references":[{"reference":"Dove, W. F., Dee, J., Hatano, S., Haugli, F. B., & Wohlfarth-Bottermann, K.-E. (Eds.). (1986). The Molecular Biology of Physarum polycephalum. Springer New York.
","pubmedId":"","doi":"10.1007/978-1-4613-2203-0"},{"reference":"Alim K, Amselem G, Peaudecerf F, Brenner MP, Pringle A. (2013). Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual. Proceedings of the National Academy of Sciences, 110(33), 13306–13311.
","pubmedId":"","doi":"10.1073/pnas.1305049110"},{"reference":"Fleischer M, Wohlfarth Bottermann KE. (1975). Correlation between tension force generation, fibrillogenesis and ultrastructure of cytoplasmic actomyosin during isometric and isotonic contractions of protoplasmic strands. Cytobiologie, 10(3), 339–365.
","pubmedId":"","doi":""},{"reference":"Giardini PA, Fletcher DA, Theriot JA. (2003). Compression forces generated by actin comet tails on lipid vesicles. Proceedings of the National Academy of Sciences, 100(11), 6493–6498.
","pubmedId":"","doi":"10.1073/pnas.1031670100"},{"reference":"Kamiya N. (1981). Physical and Chemical Basis of Cytoplasmic Streaming. Annual Review of Plant Physiology, 32(1), 205–236.
","pubmedId":"","doi":"10.1146/annurev.pp.32.060181.001225"},{"reference":"Kamiya, N. (1961). Protoplasmic streaming. Protoplasma, (53), 600-614.
","pubmedId":"","doi":"10.1007/BF01248235"},{"reference":"Kramar M, Alim K. (2021). Encoding memory in tube diameter hierarchy of living flow network. Proceedings of the National Academy of Sciences, 118(10), e2007815118.
","pubmedId":"","doi":"10.1073/pnas.2007815118"},{"reference":"Muresan CG, Sun ZG, Yadav V, Tabatabai AP, Lanier L, Kim JH, Kim T, Murrell MP. (2022). F-actin architecture determines constraints on myosin thick filament motion. Nature Communications, 13(1), 7008.
","pubmedId":"","doi":"10.1038/s41467-022-34715-6"},{"reference":"Nagai R, Yoshimoto Y, Kamiya N. (1978). Cyclic production of tension force in the plasmodial strand of Physarum polycephalum and its relation to microfilament morphology. Journal of Cell Science, 33(1), 205–225.
","pubmedId":"","doi":"10.1242/jcs.33.1.205"},{"reference":"Oettmeier C, Lee J, Dobereiner HG. (2018). Form follows function: ultrastructure of different morphotypes of Physarum polycephalum. Journal of Physics D: Applied Physics, 51(13), 134006.
","pubmedId":"","doi":"10.1088/1361-6463/aab147"},{"reference":"Quintanilla MA, Hammer JA, Beach JR. (2023). Non-muscle myosin 2 at a glance. Journal of Cell Science, 136(5), jcs260890.
","pubmedId":"","doi":"10.1242/jcs.260890"},{"reference":"Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA. (2000). Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp. The Journal of Cell Biology, 148(3), 519-530.
","pubmedId":"","doi":"10.1083/jcb.148.3.519"},{"reference":"Tilney LG, Portnoy DA. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. The Journal of Cell Biology, 109(4), 1597–1608.
","pubmedId":"","doi":"10.1083/jcb.109.4.1597"},{"reference":"Von Olenhusen KG, Wohlfarth Bottermann KE. (1979). Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell and Tissue Research, 196(3), 455–470.
","pubmedId":"","doi":"10.1007/BF00234740"}],"title":"Distinct actin architectures across a giant syncytial cell
","reviews":[],"curatorReviews":[]},{"id":"b4a315dc-f38c-4c2a-b173-882eed940509","decision":"publish","abstract":"The syncytial slime mold Physarum polycephalum exhibits cytosolic streaming, cell migration, and cell network remodeling within a single cell. It is unclear how cytoskeletal elements are spatiotemporally organized to mediate these processes. Here, we combine live and fixed imaging of Physarum F-actin to uncover organizational and dynamic signatures of the actin cytoskeleton across cell structures. We find circumferential actin filaments constrict and relax to drive peristaltic contractions in feeding tubes, while migratory fans are characterized by actin patches and comets. Our work presents undescribed actin structures in Physarum and demonstrates that different actin-based behaviors coexist within a contiguous cytoplasm.
","acknowledgements":"This work was performed at the 2023 Physiology course at the Marine Biological Laboratory (MBL), Woods Hole, directed by Dr. Nicole King and Dr. Daniel Fletcher, and in the lab of Dr. Amy Gladfelter at Duke University. We thank NIH and MBL for funding and supporting the course that made the work possible, as well as the post-course research. The course was funded by NSF grant #2014108 and Burroughs Wellcome Fund Award # 1282312, We thank Nikon and Olympus for contributing systems to the MBL Physiology course. We thank Rui Jiang and Isaac Breinyn, students of the 2023 MBL Physiology course, for capturing additional movies of Physarum polycephalum that are not included in this manuscript. We also thank the 2023 MBL Physiology course for funding the post-course research of A.C. and Z.G. We thank Dr. Christine Field and Dr. Tim Mitchison for providing LifeAct-GFP protein for injections.
","authors":[{"affiliations":["Columbia University, New York, NY, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Biomedical Engineering","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","visualization","writing_originalDraft","writing_reviewEditing"],"email":"adc2174@columbia.edu","firstName":"Andrew D.","lastName":"Countryman","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["conceptualization","investigation","methodology","writing_originalDraft","writing_reviewEditing","visualization"],"email":"zmg2@duke.edu","firstName":"Zachary M.","lastName":"Geisterfer","submittingAuthor":true,"correspondingAuthor":false,"equalContribution":true,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States"],"departments":["Department of Cell Biology"],"credit":["investigation","methodology","visualization"],"email":"christine.cao@duke.edu","firstName":"R. Christine","lastName":"Cao","submittingAuthor":false,"correspondingAuthor":false,"equalContribution":false,"WBId":null,"orcid":null},{"affiliations":["Duke University, Durham, NC, United States","Marine Biological Laboratory, Woods Hole, MA, United States"],"departments":["Department of Cell Biology","Physiology: Modern Cell Biology Using Microscopic, Biochemical and Computational Approaches"],"credit":["supervision","conceptualization","writing_reviewEditing"],"email":"amy.gladfelter@duke.edu","firstName":"Amy S.","lastName":"Gladfelter","submittingAuthor":false,"correspondingAuthor":true,"equalContribution":false,"WBId":null,"orcid":null}],"awards":[],"conflictsOfInterest":"The authors declare that there are no conflicts of interest present.
","dataTable":{"url":null},"extendedData":[{"description":"Time-lapse movies of Physarum actin dynamics in tubes (left) and fans (right), as visualized with LifeAct-GFP. The scale bar and time stamps are shared across the two movies.
","doi":null,"resourceType":"Audiovisual","name":"Extended_Data_Movie_1.mp4","url":"https://portal.micropublication.org/uploads/1a4b4b3281507c1a6d4f8c3fb8565725.mp4"}],"funding":"The Physiology course was funded by NSF grant # 2014108 and the Burroughs Wellcome Fund Award # 1282312. Z.G is supported by NIH grant # 1F32GM151858-01.
","image":{"url":"https://portal.micropublication.org/uploads/f41c89c5b8af9fb2ac1cfc922b35edd4.png"},"imageCaption":"(A) Brightfield image of the macroplasmodium of Physarum polycephalum, with the feeding tubes (blue box) and the migratory fan (red box) marked. Scale bar = 1 mm. (B) Brightfield images with inverted LUT showing the expansion of a feeding tube during peristaltic contractions. Scale bar = 50 µm. (C) Fixed, phalloidin-labeled actin structures in the feeding tube. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (D) Fixed, phalloidin-labeled actin structures in a migratory fan. Color coding indicates z-depth (see color scale bar). (E) Actin assemblies visualized through LifeAct-GFP in a live feeding tube. 3D images taken from Extended Data Movie 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (F) Actin structures, labeled with LifeAct-GFP, in a live feeding fan. 3D images taken from Extended Data Movie 1. Color coding indicates z-depth (see color scale bar). Scale bar = 50 µm. (G-I) Image of a feeding tube costained with phalloidin (magenta) and Hoechst (blue). Scale bar = 15 µm. Inset scale bar = 5 µm.
","imageTitle":"Actin ultrastructure varies across a single Physarum polycephalum macroplasmodium
","methods":"Physarum polycephalum macroplasmodia were grown on 1.7% (wt/vol) low-fluorescence water agar and fed oat flakes every two days. Twenty-four hours before imaging, 1 cm2 agar blocks containing a region of the cell were transferred to a new agar plate and left to develop a mature network in the absence of oat flakes. After starving the sample for 24 hours to reduce autofluorescence, the samples were prepared for live or fixed imaging.
For fixed imaging, sections of the network were prepared by excising an agar block containing structures of interest and gently immersing the entire agar block and specimen into 4% formaldehyde for 10-15 min at RT. After fixation, samples were rinsed three separate times using RT PBS buffer. Each rinse was approximately the volume of the initial formaldehyde immersion and was left for 5 min to allow buffer exchange within the agar block. After rinsing the specimen, samples were immersed in PBS buffer containing either 660 nM Phalloidin-Rhodamine (Fig. 1C,D) or 660 nM Phalloidin-Rhodamine and 500 nM Hoechst 33342 (Fig. 1G–I) and incubated for 10 min at RT. The specimen was rinsed once in PBS as described above, and subsequently all buffer was removed from the imaging well containing the sample. 3D fluorescence microscopy was performed on a Nikon Ti2-E inverted microscope equipped with a Yokogawa CSU-W1 spinning disk confocal. Images were acquired using a 40x Plan Apo silicon immersion objective (NA = 1.25; Fig. 1C,D). Selected images were displayed as a z-depth colored intensity projection for Fig. 1C,D. For fixed imaging of actin and nuclei, fluorescence microscopy was performed on Zeiss LSM 980 confocal. Images were acquired using a 63x Plan-Apochromat oil immersion objective (NA = 1.4; Fig. 1G-I).
For live samples, agar blocks containing regions of interest were transferred to a stereomicroscope (Zeiss Stemi 305) equipped with a FemtoJet 4i microinjector (Eppendorf). Injection needles (World Precision instruments; OD=1.0mm, ID=0.75mm) were pre-loaded with ~3 μl of LifeAct-GFP (250 µM) and set to a shallow angle of 5-10° relative to the face of the agar block. The compensation pressure was set to 60 hPa, the injection time was set to 0.5 seconds, and the injection pressure was initially set to 80 hPa. The needle was delicately introduced to a larger tube section of the specimen where the LifeAct-GFP was released. Due to the large intracellular pressure variation within and between Physarum samples, the injection pressure was increased in intervals of 10 hPa until injection resulted in visible release of LifeAct-GFP into the cell. Then, the injection was sustained until reaching a final injection volume of approximately 200 nL. Samples were left to distribute the LifeAct-GFP briefly before imaging. Time-lapse fluorescence imaging was performed on a Nikon Yokogawa CSU-W1 spinning disk confocal using a 20x air objective (Fig. 1E; Extended Data Movie 1, left) or on an Olympus IXplore SpinSR spinning disk confocal using a 20x air objective (Fig. 1F; Extended Data Movie 1, right). A single time point from the imaging is displayed as a z-depth colored intensity projection for Figure 1 E,F.
","reagents":"Phytagel (Sigma-Aldrich P8169)
Old Fashioned Oats (Quaker)
Formaldehyde (Sigma-Aldrich P47608)
1x PBS (adjust to pH 7.8 with KOH)
Rhodamine Phalloidin (Invitrogen R415)
Hoechst 33342 (ThermoFisher H1399)
Glass capillaries (World Precision instruments TW100-3)
LifeAct-GFP (250 µM)
","patternDescription":"The macroplasmodial stage of the acellular slime mold Physarum polycephalum is a syncytial, reticulated network, with millions of nuclei inhabiting a shared cytoplasm (Fig. 1; Dove et al., 1986). The cell uses long-range cytoplasmic streaming, with velocities on the order of millimeters per second, to transport bulk material across centimeter-scale intracellular distances (Fig. 1A,B; Kamiya 1961). These substantial cytoplasmic flows are driven by actomyosin-based peristaltic contractions of the cell network, although the molecular and regulatory nature of these contractions remains poorly understood (Fig. 1B; von Olenhusen & Wohlfarth-Bottermann 1979, Kamiya 1981, Alim et al., 2013). Flows transport nutrients and molecular signals across different parts of the polarized cell, from large tube structures at food sources to fan structures at migratory fronts (Fig. 1A; Kramar & Alim 2021). It remains unclear how Physarum compartmentalizes and/or coordinates different actin-based mechanical and structural behaviors across tube and fan structures within a single cytoplasm.
To visualize actin structures across Physarum macroplasmodia, we used two parallel approaches: fixed imaging of F-actin via phalloidin staining and live imaging of F-actin via injection of LifeAct-GFP. We grew macroplasmodia on fresh water-agar, then selected and excised large regions of the cell containing both fan and tube structures. These were then either microinjected with LifeAct- GFP or fixed and stained with phalloidin. Below, we characterize in more detail the distinct actin structures found in the tube and fan regions of fixed and live Physarum macroplasmodia.
First, we describe actin structures in feeding tubes. In fixed, phalloidin-labeled tubes, actin was primarily assembled into a sheet-like cell cortex and into strand-like circumferential filaments perpendicular to the long axis of the feeding tube, as previously characterized (Fig. 1C, teal; Oettmeier et al., 2018). Live imaging of tubes injected with LifeAct-GFP confirmed the presence of both sheet-like cortical (Fig. 1E, teal) and filamentous circumferential (Fig. 1E, magenta) actin structures. In these live specimens, we found that during every contraction/relaxation cycle, the circumferential filaments stretched and shortened while retaining their shape and position (Extended Data Movie 1, left). This finding differs from previous electron microscopy-based characterization of contraction-linked actin structures in Physarum, which emphasized a mechanical function of longitudinal filaments, suggested that filaments fluctuated between linear and mesh-like structures to drive each contraction cycle, and posited a role of dynamic actin filament assembly and disassembly (Nagai et al., 1978, Fleischer & Wohlfarth-Bottermann, 1975). Our fixed and live imaging clarify that circumferential, rather than longitudinal, filaments are the dominant actin structure in tubes and argue against a role of actin network disassembly or restructuring in modulating tube contraction state. This suggests that contraction may instead be controlled by regulated myosin activity, which can generate and relax tension via sliding of adjacent actin filaments without requiring complete network remodeling (Muresan et al., 2022, Quintanilla et al., 2023).
In contrast to the filamentous actin structures in tubes, fixed, phalloidin-labeled fans were instead characterized by an actin cortex with additional patch-like structures (Fig. 1D). Additionally, we observed smaller protrusive structures in both fans (Fig 1D, inset) and tubes, reminiscent of filopodia and potentially marking sites of new cell branches. Live imaging of fans injected with LifeAct-GFP revealed diffuse cortical actin as well as dense, fast-traveling actin comets (Fig. 1F; Extended Data Movie 1, right), which were also seen to a lesser extent in tubes (Extended Data Movie 1, left). In one living sample, we noticed fan remodeling and retraction away from the area illuminated by microscope light (Extended Data Movie 1, right). During this remodeling event, we observed dense actin comets associated with a transition in actin structure from a sheet-like cortical morphology into filaments (Extended Data Movie 1, right, inset). In other contexts, actin comets are known to play roles in microbe locomotion and intracellular trafficking (Tilney & Portnoy 1989, Giardini et al., 2003), but our observations here implicate them as potential mediators of actin remodeling. Together, both live and fixed imaging approaches suggest that actin structures in the fan region of the cell may be specialized to drive migration and endocytosis/exocytosis (Taunton et al., 2000) rather than cytosolic streaming, which has been attributed to the circumferential actin structures present in tubes.
To understand how nuclei were positioned with respect to the actin cytoskeleton in this syncytial cell, we co-stained fixed specimens with phalloidin and Hoechst. We found that nuclei were distributed throughout the cell, occupying phalloidin-depleted regions within an otherwise actin-rich cytoplasm (Fig. 1G–I). While it is possible that actin may play a role in nuclear positioning, we did not observe any obvious, stereotyped actin structures associated with individual nuclei. Further studies will be necessary to explore the functional importance of actin in positioning organelles under substantial cytoplasmic flow.
Overall, our work unveils and clarifies the diverse mechanostructural roles of the actin cytoskeleton in the Physarum polycephalum macroplasmodium. The whole organism contains cortical actin, which hugs the invaginated cell membrane and likely serves to position organelles and provide structural support to the cell, as well as sparse peripheral filopodial protrusions, which have not yet been described but may serve to nucleate new cell branching events. While tubes are characterized by circumferential actin filaments that constrict to drive cytoplasmic streaming, fans instead display a patch-like organization and dense actin comets, which may be associated with dynamic network remodeling, migration, and secretion. We find that actin-based structures do not significantly change or remodel across a single contraction cycle, but can convert between cortical, filamentous, and comet-like morphologies on the scale of tens of minutes. Future work probing the functions of these newly described cytoskeletal elements and uncovering the regulatory pathways that organize such distinct structures within a single cell will prove informative.
","references":[{"reference":"Alim K, Amselem G, Peaudecerf F, Brenner MP, Pringle A. (2013). Random network peristalsis in Physarum polycephalum organizes fluid flows across an individual. Proceedings of the National Academy of Sciences, 110(33), 13306–13311.
","pubmedId":"","doi":"10.1073/pnas.1305049110"},{"reference":"Dove, W. F., Dee, J., Hatano, S., Haugli, F. B., & Wohlfarth-Bottermann, K.-E. (Eds.). (1986). The Molecular Biology of Physarum polycephalum. Springer New York.
","pubmedId":"","doi":"10.1007/978-1-4613-2203-0"},{"reference":"Fleischer M, Wohlfarth Bottermann KE. (1975). Correlation between tension force generation, fibrillogenesis and ultrastructure of cytoplasmic actomyosin during isometric and isotonic contractions of protoplasmic strands. Cytobiologie, 10(3), 339–365.
","pubmedId":"","doi":""},{"reference":"Giardini PA, Fletcher DA, Theriot JA. (2003). Compression forces generated by actin comet tails on lipid vesicles. Proceedings of the National Academy of Sciences, 100(11), 6493–6498.
","pubmedId":"","doi":"10.1073/pnas.1031670100"},{"reference":"Kamiya N. (1981). Physical and Chemical Basis of Cytoplasmic Streaming. Annual Review of Plant Physiology, 32(1), 205–236.
","pubmedId":"","doi":"10.1146/annurev.pp.32.060181.001225"},{"reference":"Kamiya, N. (1961). Protoplasmic streaming. Protoplasma, (53), 600-614.
","pubmedId":"","doi":"10.1007/BF01248235"},{"reference":"Kramar M, Alim K. (2021). Encoding memory in tube diameter hierarchy of living flow network. Proceedings of the National Academy of Sciences, 118(10), e2007815118.
","pubmedId":"","doi":"10.1073/pnas.2007815118"},{"reference":"Muresan CG, Sun ZG, Yadav V, Tabatabai AP, Lanier L, Kim JH, Kim T, Murrell MP. (2022). F-actin architecture determines constraints on myosin thick filament motion. Nature Communications, 13(1), 7008.
","pubmedId":"","doi":"10.1038/s41467-022-34715-6"},{"reference":"Nagai R, Yoshimoto Y, Kamiya N. (1978). Cyclic production of tension force in the plasmodial strand of Physarum polycephalum and its relation to microfilament morphology. Journal of Cell Science, 33(1), 205–225.
","pubmedId":"","doi":"10.1242/jcs.33.1.205"},{"reference":"Oettmeier C, Lee J, Dobereiner HG. (2018). Form follows function: ultrastructure of different morphotypes of Physarum polycephalum. Journal of Physics D: Applied Physics, 51(13), 134006.
","pubmedId":"","doi":"10.1088/1361-6463/aab147"},{"reference":"Quintanilla MA, Hammer JA, Beach JR. (2023). Non-muscle myosin 2 at a glance. Journal of Cell Science, 136(5), jcs260890.
","pubmedId":"","doi":"10.1242/jcs.260890"},{"reference":"Taunton J, Rowning BA, Coughlin ML, Wu M, Moon RT, Mitchison TJ, Larabell CA. (2000). Actin-Dependent Propulsion of Endosomes and Lysosomes by Recruitment of N-Wasp. The Journal of Cell Biology, 148(3), 519-530.
","pubmedId":"","doi":"10.1083/jcb.148.3.519"},{"reference":"Tilney LG, Portnoy DA. (1989). Actin filaments and the growth, movement, and spread of the intracellular bacterial parasite, Listeria monocytogenes. The Journal of Cell Biology, 109(4), 1597–1608.
","pubmedId":"","doi":"10.1083/jcb.109.4.1597"},{"reference":"Von Olenhusen KG, Wohlfarth Bottermann KE. (1979). Evidence for actin transformation during the contraction-relaxation cycle of cytoplasmic actomyosin: cycle blockade by phalloidin injection. Cell and Tissue Research, 196(3), 455–470.
","pubmedId":"","doi":"10.1007/BF00234740"}],"title":"Distinct actin architectures across a giant syncytial cell
","reviews":[],"curatorReviews":[]}]}},"species":{"species":[{"value":"acer saccharum","label":"Acer saccharum","imageSrc":"","imageAlt":"","mod":"TreeGenes","modLink":"https://treegenesdb.org","linkVariable":""},{"value":"achillea millefolium","label":"Achillea millefolium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"acinetobacter baylyi","label":"Acinetobacter baylyi","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"actinobacteria bacterium","label":"Actinobacteria bacterium","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adelges tsugae","label":"Adelges tsugae","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"adenocaulon chilense","label":"Adenocaulon chilense","imageSrc":"","imageAlt":"","mod":"","modLink":"","linkVariable":""},{"value":"aedes japonicus","label":"Aedes 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