Description
The fission yeast
Schizosaccharomyces pombe
utilizes an actin- and myosin-based cytokinetic ring (CR) to accomplish cytokinesis (Cheffings et al., 2016; Glotzer, 2017; Mangione & Gould, 2019). The
S. pombe
CR is built from medial cytokinetic nodes established by the anillin-like
Mid1
protein (reviewed in Rincon & Paoletti, 2012). These nodes condense into a coherent ring structure that will eventually constrict concomitant with septum ingression (reviewed in Willet et al., 2015). The precise spatial and temporal ordering of mitotic and cytokinetic events is critical for the faithful segregation of the genetic material into daughter cells. One mechanism by which mitosis and cytokinesis are coordinated is through the action of mitotic kinases that phosphorylate substrates in a cell cycle-dependent manner. One such enzyme is the
S. pombe
polo kinase
Plo1
which operates downstream of Cdk1 to promote successful mitosis and cytokinesis (Ohkura et al., 1995; Tanaka et al., 2001). One of
Plo1
's substrates is
Mid1
and Plo1-dependent phosphorylation of
Mid1
promotes the nuclear export of
Mid1
(Almonacid et al., 2011; Bähler et al., 1998; Ohkura et al., 1995). This ensures that
Mid1
can localize to the medial cytokinetic nodes at mitotic entry to promote CR formation (Almonacid et al., 2009, 2011). Here, we examine
mid1
and
plo1
temperature-sensitive alleles obtained from various genetic screens, some previously uncharacterized and many lacking information as to the sites of mutations (Bähler et al., 1998; Balasubramanian et al., 1998; MacIver et al., 2003).
To better understand the nature of the
mid1
and
plo1
alleles, the respective open reading frames were amplified from nine strains (
mid1-18, mid1-C1,
mid1-C2, mid1-2075, plo1-1, plo1-ts4, plo1-24c, plo1-25
and
plo1-ts35
) in which the relevant mutations have not been reported (Rutherford et al., 2024) and the PCR products sequenced. In each case, a single point mutation was detected (
Figure 1A
).
mid1-18
encodes a serine to phenylalanine substitution at position 873 (S873F), both
mid1-C1
and
mid1-C2
encode a glycine to aspartic acid substitution at position 764 (G764D) and
mid1-2075
encodes a glycine to aspartic acid substitution at position 584 (
Figure 1A
). All of the mutated amino acids reside in structured regions of the protein (
Figure 1A
). Another previously characterized
mid1
allele,
mid1-366
, encodes a different mutation within the C2 domain (G718D) (Chang et al., 1996; Sun et al., 2015). Thus, our results bring the total number of distinct
mid1
temperature-sensitive alleles to four.
Two
plo1
alleles (
plo1-1
and
plo1-ts4)
encode the same mutation causing an arginine to glutamine change at residue 132 (R132Q).
plo1-24c
encodes a mutation resulting in a change at residue 295 from phenylalanine to leucine,
plo1-25
encodes an arginine to tryptophan substitution at residue 141 (R141W) and
plo1-ts35
encodes a glutamic acid to lysine change at position 139 (E139K) (
Figure 1A
). E139K is the same mutation previously identified in the
plo1-ts2
allele (MacIver et al., 2003). Thus, in addition to two previously sequenced temperature-sensitive
plo1
alleles (
plo1-ts2
and
plo1-ts19
), there are three additional distinct temperature-sensitive
plo1
alleles.
To examine the
mid1
and
plo1
mutant cohorts further, we compared the temperature-sensitivity of each strain with a growth assay.
mid1-18, mid1-C1 and mid1-C2
grew similar to wildtype at all temperatures but
mid1-2075
showed almost no growth at 36°C (
Figure 1B
). Two
plo1
alleles,
plo1-ts2
and
plo1-ts35,
had almost no growth at 36°C and reduced growth at 32°C, and all other
plo1
alleles were slightly impaired in growth compared to wildtype at 36°C (
Figure 1B
). These results are consistent with previously published growth assays in which some of the mutants were examined (MacIver et al., 2003; Petersen & Hagan, 2005; Rachfall et al., 2014).
Finally, to visualize and compare the cell phenotypes, we examined each mutant by staining for nuclei and septa after the cells were grown at 25°C and then shifted to 36°C for 4 hours. We found that all temperature sensitive alleles
looked similar to wildtype at 25°C, but at 36°C the cells were multinucleated with abnormal septa present (
Figure 1C-
D). Our data are consistent with previous descriptions of the subset of previously studied alleles (Bähler et al., 1998; Balasubramanian et al., 1998; Bhutta et al., 2014; Huang et al., 2008; MacIver et al., 2003; Wachtler et al., 2006).
All
mid1
temperature-sensitive alleles displayed phenotypes similar to
mid1∆
(Chang et al., 1996; Sohrmann et al., 1996), suggesting that they are loss-of-function alleles.
mid1-18
has a mutation in the PH domain while
mid1-C1
,
mid1-C2, mid1-2075
and
mid1-366
contain mutations in the C2 domain (
Figure 1A
). The C2 domain appears to directly bind the plasma membrane and is required for
Mid1
function (Lee & Wu, 2012; Sun et al., 2015). Cells with a mutant
Mid1
lacking the entire PH domain have only mild cell division site positioning defects, thus the function of the PH domain is not fully clear (Lee & Wu, 2012; Paoletti & Chang, 2000). It has been suggested that the PH domain may be important for protein stability and/or play a role in membrane binding in collaboration with the C2 domain, like human anillin (Hall et al., 2024; Liu et al., 2012; Sun et al., 2015). Taken together, we speculate that the
mid1-18
allele produces a less stable protein at high temperatures while the other alleles disrupt C2 domain function.
Plo1
has two essential domains, the N-terminal kinase domain and the C-terminal polo box domain (PBD) (Reynolds & Ohkura, 2003) (
Figure 1A
). While the kinase domain phosphorylates protein substrates, the PBD binds both substrates and protein partners to direct subcellular localization (Park et al., 2010). Interestingly, all mutations encoded by the
plo1
temperature sensitive alleles map within the kinase domain and none are within the PBD (
Figure 1A
). The one unique allele previously sequenced is
plo1-ts19
which contains an early stop codon at tryptophan 316 (MacIver et al., 2003). The W316* mutation truncates an ɑ-helix within the kinase domain and eliminates an unstructured region and the PBD (MacIver et al., 2003) which is a surprising result given the reported requirement for the PBD for
Plo1
function (Reynolds & Ohkura, 2003).
To follow up these observations, we used AlphaFold3 to model the
Plo1
kinase domain with an ATP molecule (Abramson et al., 2024), and mapped the mutated residues encoded by the
plo1
alleles onto the predicted structure. We found that all of the mutated residues clustered in the C-terminal lobe of the kinase domain (
Figure 1E
). Deciphering if ATP binding, substrate binding or other protein partner binding is defective in these
plo1
alleles will be exciting direction for future studies. Further, how cells tolerate a lack of the
Plo1
PBD will be an interesting avenue of study.
Methods
Yeast methods
S. pombe
strains were grown in yeast extract (YE) and standard
S. pombe
mating, sporulation, and tetrad dissection techniques were used to construct new strains (Moreno et al., 1991).
Molecular biology methods
The
mid1
open reading frames from
mid1-C1
and
mid1-C18
cells were amplified by generating a PCR product with an oligonucleotide
74 bp upstream of the start site (GTTGTACTTCAGGGTGCTTA) and an oligonucleotide 380 bp downstream of the stop codon (AGGTTCTCCATCTCATGGCT) (Integrated DNA technologies). The
mid1
open reading frame was amplified from
mid1-C2
cells using two sets of overlapping oligonucleotides. The first PCR product was generated with the above-mentioned oligonucleotide 74 bp upstream of the start site and an oligonucleotide 1350 bp into the open reading frame (GTTGCATTGATGGGTGACGT). The second PCR product was produced with an oligonucleotide 1200 bp within the open reading frame (GTATGGTCATGGATCTGTAACG) and the above-mentioned oligonucleotide 380 bp downstream of the stop codon.
The
plo1
alleles were amplified by generating two PCR products. One PCR reaction used an oligonucleotide 40 bp upstream of the start site (GCAACCACTTTGTTTACCCTCA) and an oligonucleotide 1100 bp into the open reading frame of
plo1
(TGGACTTAAAACACTTGGTAATATTCG) (Integrated DNA technologies). The second PCR reaction used an oligonucleotide 900 bp into the open reading frame (TCCAGATGAAATTTTACATTCAATGCCT) with an oligonucleotide 20 bp downstream of the stop codon (GCATAGTAACTTAACGCCCAAGTA).
The PCR products were sequenced by Plasmidsaurus using Oxford Nanopore Technology with custom analysis and annotation.
Microscopy and image analysis
Strains for fixed-cell imaging experiments were grown at 25°C in YE and then shifted to 36°C for 3 hours. Cells were fixed with 70% ethanol for DAPI and methyl blue (MB) staining as described previously (Roberts-Galbraith et al., 2009). Images were acquired using a Zeiss Axio Observer inverted epifluorescence microscope with Zeiss 63× oil (1.46 NA) objective and captured using Zeiss ZEN 3.0 (Blue edition) software. A singular medial Z slice was obtained. All images were further processed using ImageJ (Schindelin et al., 2012).
AlphaFold3 structural prediction
Protein structure predictions were generated with the AlphaFold3 server (Abramson et al., 2024) and visualized using the PyMOL molecular graphics system (version 3.0, Schrodinger, LLC).