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View Code? Open in Web Editor NEWA repository for more broad discussion of curation
License: Creative Commons Attribution Share Alike 4.0 International
A repository for more broad discussion of curation
License: Creative Commons Attribution Share Alike 4.0 International
Need to discuss active form usage with Val. We requested a term and we may be able to use it for noctua if all logical definitions are correct. Now the session is approved, will have to be re-opened if we go for this.
Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1-385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1's kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover. IMPORTANCE Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP8 synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP6. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP8 is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP8 turnover.
IP7 and IP8 are singaling molecules involved in eukaryal phosphate homeostasis -> transcriptional response to phosphate availability, upregulates genes involved in extracellular phosphate acquisition.
IP6 -Kcs1-> 5-IP7 -Asp1-> 1,5-IP8
Asp1 is a bifunctional enzyme:
Isolated N-term has the activity
Fission yeast phosphate homeostasis (PHO) regulon three phosphate acquisition genes:
Reressed by flancking lncRNAs prt, prt2 and nc-tgp1
asp1 that cannot do pirophospatase reaction (asp1-H397A) increases IP8 levels and derepresses PHO regulon -> termination of prt lncRNA synthesis.
asp1 deletion (no IP8) -> pho1 hyperrepression
asp1 syntehtic lethal with CPF subunits mutations: maybe IP8 or IP7 important for 3'-processing / termination events (agonist of pol2 transc termination)
Cells that are asp1D and express asp1(1-385) mutants from a plasmid, tgp1 promoter, and plasmid bears a nc-tgp1 lncRNA under nmt1 promoter.
The assay measures the Pho1 acid phosphatase activity in cells:
strong preference of Asp1 kinase for 5-IP7 versus IP6
The experiment quantifies the acid phosphatase activity activity, pressumably of Pho1 (regulated by the PHO regulon). Mutations in asp1 enzyme can lead to the activation/repression of pho1 expression, and therefore high/low enzymatic activity. The authors have annotated:
However, this is not really what was measured, but rather the pho1 activity. At the same time, I am not sure a molecular function phenotype is correct either, since these phenotypes are probably to indicate that enzymatic activity is altered at the molecule level, not because there is more / less enzyme.
I guess in this case what makes more sense is to use "increased / decreased protein level" for pho1?
Microtubules (MTs) and associated motors play a central role in nuclear migration, which is crucial for diverse biological functions including cell division, polarity, and sexual reproduction. In this paper, we report a dual mechanism underlying nuclear congression during fission yeast karyogamy upon mating of haploid cells. Using microfluidic chambers for long-term imaging, we captured the precise timing of nuclear congression and identified two minus end-directed motors operating in parallel in this process. Kinesin-14 Klp2 associated with MTs may cross-link and slide antiparallel MTs emanating from the two nuclei, whereas dynein accumulating at spindle pole bodies (SPBs) may pull MTs nucleated from the opposite SPB. Klp2-dependent nuclear congression proceeds at constant speed, whereas dynein accumulation results in an increase of nuclear velocity over time. Surprisingly, the light intermediate chain Dli1, but not dynactin, is required for this previously unknown function of dynein. We conclude that efficient nuclear congression depends on the cooperation of two minus end-directed motors.
Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B
Deletion of several genes does not prevent microtubule growth speed decrease during anaphase, although they may affect the actual speed.
Genotype | Phenotype | Figure |
---|---|---|
cls1 overexp | reduced growth speed during anaphase | Fig. 2 supp 2F |
cls1 shutdown | increased growth speed during anaphase | Fig. 2 supp 2E |
mal3D | reduced growth speed during anaphase | Fig. 2H |
klp5Dklp6D | increase duration of mt growth event / reduced catastrophe | Fig. 2 supp 1 |
Genotype | Phenotype | Figure |
---|---|---|
Klp9OE overexp | Longer cells / faster spindle elongation anaphase B | Fig. 3 supp 1 A, C |
cdc25-22 | Longer cells / faster spindle elongation anaphase B | Fig. 3 supp 1 A, C |
These were already published in Kruger et al. 2019, so I guess they should be included when that paper is added. Except longer cells in Klp9OE, son include that one.
https://pubmed.ncbi.nlm.nih.gov/30806623/
Genotype | Phenotype | Figure |
---|---|---|
les1D | Faster MT growth in nuclear membrane bridge | Fig. 4 supp 2 |
nem1D | Faster MT growth in nuclear membrane bridge | Fig. 4 supp 2 |
Genotype | Phenotype | Figure |
---|---|---|
ase1D | Loss of microtubule rescue organisation | Fig. 5E |
ase1D | Faster MT growth in nuclear membrane bridge | Fig. 5F,G |
ase1 shutdown | increased growth speed during anaphase | Fig. 5 supp 3E |
Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination.
NOTE:
rec10D does not form LinEs, while rec8 forms aberrant LinEs. Why rec12?
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
rec8D | Decreased alignment index | Fig. 2A, B |
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
rec8D | Decreased loops | Fig. 3C | |
rec10D | A bit decreased loops? | Fig. 3C |
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
9,10 how to tell the difference with strong recruitment? | wpl1D | Increased recruitment of Rec8-GFP to the structures during horsetail + more obvious elongated structure rather than fuzzy (suggesting that the axial structure becomes thicker) | Fig. 4A |
8, but check specificity of term | wpl1D | Reduced chromosome pairing during horsetail | Fig. S4B |
11 | wpl1D | Longer loops | Fig. 4B |
wpl1D | Reduced chromosome pairing in meiosis and VG (see how the centromere connexion is the same actually) | Fig. S4B | |
wpl1Drec10D | No LinEs (probably not worth curating since it's like rec10D) | Fig. S4F | |
wpl1Drec10D | Formation of thick elements (meaning that formation of LinEs is not required for this thick structures) | Fig. S4F |
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
rec8-F204S + background | Normal cell growth | Fig. S5B | |
rec8-F204S + background | No formation of thick structures, but cohesin activity | Fig. 5B | |
3, but mei4 maybe should be added? | rec8-F204S + background only mei4 and locus tagged | No change in sister chromatid pairing. This is an assay for cohesin activity, no horsetail! | Fig. 5C |
6 | rec8-F204S no background | Phenotype in which the spb moves with horsetail but not the rest. Not entirely sure what the difference between deletion and mutant are | Fig. 5D |
rec8-F204S no background | Stretched telomere - ade8 distance. Like deletion | Fig. 5E |
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
rec8-F204S | Lower big loop formation Hi-C as deletion? looks like it | Fig. 6A | |
4 | rec8-F204S | Lower small loop formation Hi-C lower severity than deletion | Fig. 6A |
rec8-F204S | Lower recruitment to known Rec8-enriched sites of cohesin rec8 (ChIP) | Fig. S6B | |
rec8-F204S | Normal recruitment to other genome positions (ChIP) | Fig. S6B |
Row in curs table | Genotype | Phenotype | Figure |
---|---|---|---|
1 | rec8-F204S | aberrant LinEs as seen with Rec10-mCherry | Fig. 7A |
2,5? | rec8-F204S | Lower recombination (known consequence of aberratn LinEs) | Fig. 7B |
rec8-F204S | Normal phosphorylation of Rec11 (not to curate I guess) | Fig. 7C |
Mitosis is a fundamental process in every eukaryote, in which chromosomes are segregated into two daughter cells by the action of the microtubule (MT)-based spindle. Despite this common principle, genes essential for mitosis are variable among organisms. This indicates that the loss of essential genes or bypass of essentiality (BOE) occurred multiple times during evolution. While many BOE relationships have been recently revealed experimentally, the bypass of essentiality of mitosis regulators (BOE-M) has been scarcely reported, and how this occurs remains largely unknown. Here, by mutagenesis and subsequent evolutionary repair experiments, we isolated viable fission yeast strains that lacked the entire coding region of Polo-like kinase (Plk), a versatile essential mitotic kinase. The BOE of Plk was enabled by specific mutations in the downstream machinery, including the MT-nucleating γ-tubulin complex, and more surprisingly, through down-regulation of glucose uptake, which is not readily connected to mitosis. The latter bypass was dependent on casein kinase I (CK1), which has not been considered as a major mitotic regulator. Our genetic and phenotypic data suggest that CK1 constitutes an alternative mechanism of MT nucleation, which is normally dominated by Plk. A similar relationship was observed in a human colon cancer cell line. Thus, our study shows that BOE-M can be achieved by simple genetic or environmental changes, consistent with the occurrence of BOE-M during evolution. Furthermore, the identification of BOE-M constitutes a powerful means to uncover a hitherto understudied mechanism driving mitosis and also hints at the limitations and solutions for selecting chemotherapeutic compounds targeting mitosis.
To find suppressors of essential genes, they make heterozygous deletion in a diploid, sporulate, irradiate the spores with UV, grow the spores in the selective medium for the deletion resistance marker, sequence the colonies obtained.
plo1D: inviable
plo1D ght5D: viable
plo1D viable 0.08% glucose
plo1 and:
ght5D
ght5D plo1D
hhp1 1% monopolar spindle
hhp1 hhp2 29% monopolar spindle
bub1Δ ght5Δ plo1Δ, anaphase began even when spindles were still monopolar in 13 of 43 cell
hhp1Δ ght5Δ plo1Δ, >80% of cells were arrested in monopolar states for >30 min
Microtubule (MT) arrays are mechanistic effectors of polarity specification and cell division. Linear bundles in which MTs are bridged laterally are dynamically assembled in systems ranging from differentiated metazoan cells to fungi in a process that remains poorly understood. Often, bundled MTs slide with respect to each other via molecular motors. In interphase cells of the fission yeast Schizosaccharomyces pombe, MT nucleation frequently occurs at preexisting arrays. As the nascent MT lengthens, stable antiparallel MT overlaps are thought to form through competition between motion of the minus-end-directed kinesin Klp2p and braking force exerted by the accumulating lateral crosslinker Ase1p. Here we show that Mia1p/Alp7p, a transforming acidic coiled-coil (TACC) protein, functions as a length-independent MT crosslinker. In cells lacking Mia1p MT-bundling activity, linear arrays frequently disassemble, accompanied by a marked increase in Ase1p off rate and erratic motion of sliding MTs. We propose that the combined action of lateral length-dependent (Ase1p) and terminal length-independent (Mia1p) crosslinkers is crucial for robust assembly and stability of linear MT arrays. Such use of qualitatively distinct crosslinking mechanisms in tandem may point to a general design principle in the engineering of stable cytoskeletal assemblies.
Alp7 localizes to the plus ends of microtubules
Alp7 localizes to the edges of MT overlaps
Partial colocalization with mto1
Partial colocalization with Tip1
Purified Alp7 bundles microtubules in vitro (68% antiparallel). Moved to #18
Mia1p-1-300-GFP:
Mia1p-1-300-GFP in pim1-1 background
Mia1p-301-474-GFP)
MBP-Mia1p-D151-300
Antagonistic Spindle Motors and MAPs Regulate Metaphase Spindle Length and Chromosome Segregation
Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at characteristic constant length [1-3]. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules (MTs) and their interactions with motors and MT-associated proteins (MAPs). Spindle length is further proposed to be important for chromosome segregation fidelity, as cells with shorter- or longer-than-normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force-balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature control with live-cell imaging to monitor the effect of deleting or switching off different combinations of antagonistic force contributors in the fission yeast metaphase spindle. We show that the spindle midzone proteins kinesin-5 cut7p and MT bundler ase1p contribute to outward-pushing forces and that the spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward-pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and in some combinations also partially rescued chromosome segregation defects.
Longer spindle:
Shorter spindle:
Normal spindle:
Increased spindle elongation speed during phase I (prophase)
Reduced spindle elongation speed during phase I (prophase)
Increased time before anaphase (increased duration of Phase I and II combined)
Rescued duration
Partial rescue of metaphase length
Total rescue of metaphase length
Rescued duration
In the text only
Spindle collapse after normal metaphase spindle formation (Fig. 2A-C). There may be an instance where shifting to the restrictive temperature would not cause collapse, this experiment shows that cut7 is not only required to stablish bipolarity, but also to maintain it.
No spindle collapse (after normal metaphase spindle formation)??
Hi @ValWood I am annotating now klp2 cellular component to microtubule plus ends. Within GO, can we indicate that this localization depends on another protein (in this case mal3), or does this belong only to the phenotype ontologies?
To be discussed at some point
Proteins related to the phosphoinositide-dependent protein kinase family have been identified in the majority of eukaryotes. Although much is known about upstream mechanisms that regulate the PDK1-family of kinases in metazoans, how these kinases regulate cell growth and division remains unclear. Here, we characterize a fission yeast protein related to members of this family, which we have termed Pdk1p. Pdk1p localizes to the spindle pole body and the actomyosin ring in early mitotic cells. Cells deleted for pdk1 display multiple defects in mitosis and cytokinesis, all of which are exacerbated when the function of fission yeast polo kinase, Plo1p, is partially compromised. We conclude that Pdk1p functions in concert with Plo1p to regulate multiple processes such as the establishment of a bipolar mitotic spindle, transition to anaphase, placement of the actomyosin ring and proper execution of cytokinesis. We also present evidence that the effects of Pdk1p on cytokinesis are likely mediated via the fission yeast anillin-related protein, Mid1p, and the septation initiation network. © 2005 by The American Society for Cell Biology.
pdk1D (background cdc25-22)
pdk1D mph1D (background cdc25-22)
At normal temp
pdk1D
mid1D cells don't localize pdk1D to the cortical center (site of cell division) -> depends on annotation!
pdk1D sid2-250, pdk1D spg1-106, pdk1D sid4-A1 were unable to divide and form colonies at all
Hi Manu,
As you will see I have assigned you lots of sessions. I think most of these will be relatively quick for curation; some, especially the Tolic ones will probably only have one or two GO terms, but these are useful because describing normal functions and processes of genes is one of our main aims.
For example this one
https://core.ac.uk/reader/33995484?utm_source=linkout
we can add GO process "nuclear positioning" fir kinesin-8 motors
currently GO:0007097 nuclear migration (exact synonym).
Nuclear positioning seems slightly broader to than migration to me and should be the exact synonym (or even primary term name) of the parent "nucleus localization", do you agree?
Pending FYPO: pombase/fypo#4164
Aneuploidy - chromosome instability leading to incorrect chromosome number in dividing cells - can arise from defects in centrosome duplication, bipolar spindle formation, kinetochore-microtubule attachment, chromatid cohesion, mitotic checkpoint monitoring or cytokinesis. As most tumours show some degree of aneuploidy, mechanistic understanding of these pathways has been an intense area of research, to provide potential therapeutics. Here we present a mechanism for aneuploidy in fission yeast based on spindle pole microtubule defocusing by loss of kinesin-14 Pkl1, leading to kinesin-5 Cut7-dependent aberrant long spindle microtubule minus-end protrusions that push the properly segregated chromosomes to the site of cell division, resulting in chromosome cut at cytokinesis. Pkl1 localization and function at the spindle pole is mutually dependent on spindle pole-associated protein Msd1. This mechanism of aneuploidy bypasses the known spindle assembly checkpoint that monitors chromosome segregation.
I wonder if "long" is necessary in FYPO:0003787. This paper goes in detail and show that both minus end long-lived and plus end short-lived protrusions are formed.
More detailed here:
They show that there are two kinds of protrusions in the spindle.
In PMID:26031557 they show that removing cut7 (the kinesin that slides microtubule during metaphase) gets rid of the long lived protrusions, but not the short ones. The interpretation here would be that:
Create ticket in pombase/curation.
Create ticket for FYPO:0004087.
Probably the GO-term GO:0034631 microtubule anchoring at spindle pole body could have a parent that is spindle pole organisation or something like that. Kinesin-14 should have shared function in certain cell types with the pkl1 phenotype. Worth a discussion probably
Go ticket
In pombe, unless using EM what is the difference between cellular component spindle pole (GO:0097431)and cellular component spindle pole body (GO:0044732). Also, ase1 is included there but it's actually outside (would have to look for the reference where they show this but likely Toda ase1 paper, or Loiodice).
pombase/curation#3250 with val
There are currently the following terms referring to microtubule dynamics, with their children:
graph LR;
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0007706[FYPO:0007706<br>decreased microtubul<br>e depolymerization a<br>t cell side];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0004650[FYPO:0004650<br>decreased mitotic sp<br>indle microtubule de<br>polymerization];
FYPO:0004650[FYPO:0004650<br>decreased mitotic sp<br>indle microtubule de<br>polymerization]-->FYPO:0005423[FYPO:0005423<br>decreased mitotic sp<br>indle microtubule de<br>polymerization durin<br>g anaphase A];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0004616[FYPO:0004616<br>abolished cytoplasmi<br>c microtubule depoly<br>merization at plus e<br>nd at cell tip];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0003194[FYPO:0003194<br>increased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0005681[FYPO:0005681<br>decreased microtubul<br>e polymerization];
FYPO:0000901[FYPO:0000901<br>abnormal microtubule<br> polymerization or d<br>epolymerization duri<br>ng vegetative growth]-->FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0007182[FYPO:0007182<br>decreased cytoplasmi<br>c microtubule depoly<br>merization at plus e<br>nd at cell tip];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0005682[FYPO:0005682<br>decreased microtubul<br>e depolymerization d<br>uring vegetative gro<br>wth];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0003190[FYPO:0003190<br>decreased rate of cy<br>toplasmic microtubul<br>e depolymerization];
FYPO:0003194[FYPO:0003194<br>increased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth]-->FYPO:0004976[FYPO:0004976<br>increased rate of mi<br>crotubule depolymeri<br>zation ahead of movi<br>ng spindle pole body];
FYPO:0003194[FYPO:0003194<br>increased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth]-->FYPO:0004617[FYPO:0004617<br>increased rate of mi<br>crotubule depolymeri<br>zation at minus end];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0005703[FYPO:0005703<br>decreased rate of mi<br>crotubule polymeriza<br>tion];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0004615[FYPO:0004615<br>increased rate of in<br>terphase microtubule<br> polymerization];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0000903[FYPO:0000903<br>decreased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0006180[FYPO:0006180<br>increased microtubul<br>e polymerization];
FYPO:0000903[FYPO:0000903<br>decreased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth]-->FYPO:0004721[FYPO:0004721<br>decreased rate of mi<br>crotubule depolymeri<br>zation at minus end];
FYPO:0004650[FYPO:0004650<br>decreased mitotic sp<br>indle microtubule de<br>polymerization]-->FYPO:0005363[FYPO:0005363<br>decreased kinetochor<br>e microtubule depoly<br>merization during mi<br>totic metaphase chro<br>mosome recapture];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0005463[FYPO:0005463<br>decreased spatial ex<br>tent of interphase m<br>icrotubule polymeriz<br>ation];
FYPO:0000901[FYPO:0000901<br>abnormal microtubule<br> polymerization or d<br>epolymerization duri<br>ng vegetative growth]-->FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization];
FYPO:0000903[FYPO:0000903<br>decreased rate of mi<br>crotubule depolymeri<br>zation during vegeta<br>tive growth]-->FYPO:0004975[FYPO:0004975<br>decreased rate of mi<br>crotubule depolymeri<br>zation behind moving<br> spindle pole body];
FYPO:0000902[FYPO:0000902<br>abnormal microtubule<br> depolymerization du<br>ring vegetative grow<br>th]-->FYPO:0006105[FYPO:0006105<br>increased microtubul<br>e depolymerization a<br>t cell side];
FYPO:0004614[FYPO:0004614<br>abnormal microtubule<br> polymerization]-->FYPO:0006067[FYPO:0006067<br>abolished microtubul<br>e polymerization];
graph LR;
FYPO:0007111[FYPO:0007111<br>abnormal microtubule<br> depolymerization]-->FYPO:0007112[FYPO:0007112<br>decreased rate of mi<br>crotubule depolymeri<br>zation involved in m<br>eiotic centromere cl<br>ustering during meio<br>tic prophase I];
These two should be probably add "during vegetative growth", since they are children of "FYPO:0000899", "normal microtubule cytoskeleton organization during vegetative growth "
Then there is "abnormal kinetochore microtubule polymerization during mitotic metaphase chromosome recapture" FYPO:0006040, which should also be linked to mt dynamics, and is only linked to FYPO:0005362 (abnormal mitotic metaphase chromosome recapture)
Rename FYPO:0000901 to abnormal microtubule dynamics during vegetative growth. Then make four children:
The annotations from terms that are not specific and just say "decrease depolymerisation" should be revisited and assigned to the right term (depending on whether they affected shrinkage speed or growth event duration). Same applies to others like FYPO:0005463 (decreased spatial extent of interphase microtubule polymerization), they probably can be pinpointed to increased/decreased growth/shrinkage speed or duration. I can do this in the future.
Rename FYPO:0007111 to depol. rate, since the child is specific to that
Adding "during vegetative growth" to FYPO:0003193 and FYPO:0003225, since they are children of "FYPO:0000899", "normal microtubule cytoskeleton organization during vegetative growth "
Linking FYPO:0006040 to the child of FYPO:0000901 with decrease microtubule depolymerisation rate.
Edit the synonyms as well.
Term name/ id | Request | New definition | New name | Child of (if added) |
---|---|---|---|---|
FYPO:0000901 | Change name and definition | A cellular process phenotype observed in the vegetative growth phase of the life cycle in which microtubule dynamics are abnormal. | abnormal microtubule dynamics during vegetative growth | |
Abnormal microtubule depolymerisation rate | Addition | A cellular process phenotype observed in the vegetative growth phase of the life cycle in which the rate, or speed, of microtubule polymerization, i.e. the addition of tubulin dimers to a microtubule, is abnormal (matching FYPO:0003225) | FYPO:0000901 | |
Abnormal microtubule polymerisation rate | Addition | A cellular process phenotype observed in the vegetative growth phase of the life cycle in which the rate, or speed, of microtubule depolymerization, i.e. the removal of tubulin dimers to a microtubule, is abnormal (matching FYPO:0003193) | FYPO:0000901 | |
Abnormal microtubule growth event duration | Addition | A cellular process phenotype observed in the vegetative growth phase of the life cycle in which the duration of microtubule growth events is abnormal | FYPO:0000901 | |
Abnormal microtubule rescue | Addition | A cellular process phenotype observed in the vegetative growth phase of the life cycle in which microtubule rescue (the transition from shrinkage to growth) is abnormal. The frequency of rescues as well as the organisation can be affected. | FYPO:0000901 | |
FYPO:0007111 | Change name and definition | A cellular process phenotype in which the rate, or speed, of microtubule polymerization, i.e. the addition of tubulin dimers to a microtubule, is abnormal (matching FYPO:0003225) | Abnormal microtubule depolymerization rate |
These are either for FYPO:0000901, or for the terms that were already specific and mentioned growth/shrinkage rate.
Term name/ id | Existing synonym | Proposed change | Reason |
---|---|---|---|
FYPO:0000901 | all | None | I guess they can be left like this, since they mention both growth and shrinkage |
FYPO:0003193 | normal rate of microtubule catastrophe | remove | Catastrophe, not depolymerisation. |
FYPO:0003194 | The ones that mention stability and catrastrophe | remove | Catastrophe, not depolymerisation. |
FYPO:0003190 | The ones that mention stability and catrastrophe | remove | Catastrophe, not depolymerisation. |
FYPO:0007111 | catastrophe | remove | Catastrophe, not depolymerisation. |
Bipolar spindle assembly requires a balance of forces where kinesin-5 produces outward pushing forces to antagonize the inward pulling forces from kinesin-14 or dynein. Accordingly, Kinesin-5 inactivation results in force imbalance leading to monopolar spindle and chromosome segregation failure. In fission yeast, force balance is restored when both kinesin-5 Cut7 and kinesin-14 Pkl1 are deleted, restoring spindle bipolarity. Here we show that the cut7Dpkl1D spindle is fully competent for chromosome segregation independently of motor activity, except for kinesin-6 Klp9, which is required for anaphase spindle elongation. We demonstrate that cut7Dpkl1D spindle bipolarity requires the microtubule antiparallel bundler PRC1/Ase1 to recruit CLASP/Cls1 to stabilize microtubules. Brownian dynamics- kinetic Monte Carlo simulations show that Ase1 and Cls1 activity are sufficient for initial bipolar spindle formation. We conclude that pushing forces generated by microtubule polymerization are sufficient to promote spindle pole separation and the assembly of bipolar spindle in the absence of molecular motors.
And annotated proteins vs SPB, such as motors etc.
Bipolar spindle assembly is a critical control point for initiation of mitosis through nucleation and organization of spindle microtubules and is regulated by kinesin-like proteins. In fission yeast, the kinesin-14 Pkl1 binds the g-tubulin ring complex (g-TuRC) microtubule-organizing centre at spindle poles and can alter its structure and function. Here we show that kinesin-14 blocks microtubule nucleation in yeast and reveal that this inhibition is countered by the kinesin-5 protein, Cut7. Furthermore, we demonstrate that Cut7 binding to g-TuRC and the Cut7 BimC domain are both required for inhibition of Pkl1. We also demonstrate that a yeast kinesin-14 peptide blocks microtubule nucleation in two human breast cancer cell lines, suggesting that this mechanism is evolutionarily conserved. In conclusion, using genetic, biochemical and cell biology approaches we uncover antagonistic control of microtubule nucleation at g-TuRC by two kinesin-like proteins, which may represent an attractive anti-mitotic target for cancer therapies.
Co-IPs
cut7 in gtb1-k5a background:
cut7HS in gtb1-k5a background:
cut7ST in gtb1-k5a background:
NLS-Cut7ST localizes to SPBs only, not to spindle microtubules. This is quite important although already mentioned in Hagan paper
NLS-Cut7T localizes to SPBs only, not to spindle microtubules.
NLS-Cut7T22 all localization lost.
NLS-Cut7ST22 all localization lost.
Revise refs 17, 22, 23 -> Interaction of pkl1 with gamma-tub
Ref. 23 -> pkl1 replaces HSET in human.
all human g-TuSC protein components are also compatible.
Hagan paper nucleolus -> nucleus in the localization phenotype.
Annotations from Yukawa paper to FYPO:0006339 are a bit confusing. The mislocalized nucleus annotation should maybe replaced by only by diploidisation, and the ones that don't do this by cut phenotype (if curated at all since these are downstream effects of failure to form a spindle). What happens is that the nucleus does not divide and it's pushed to one of the daughter cells. Also some annotations for the Hagan paper are here.
Revise delayed onset of anaphase B, and ana/meta transition. There might be an issue on this already.
Cut7 allele annotations from Hagan's paper, they are the same here:
Name | Aminoacids kept |
---|---|
cut7-ST | 443-1085 |
cut7-H | 1-443 |
cut7-T | 888-1085 |
cut7-HS | 1-888 |
cut7-22 | Pro1021->Ser |
cut7ST-22 | Pro1021->Ser |
cut7T-22 | Pro1021->Ser |
The telomerase holoenzyme is critical for maintaining eukaryotic genome integrity. In addition to a reverse transcriptase and an RNA template, telomerase contains additional proteins that protect the telomerase RNA and promote holoenzyme assembly. Here we report that the methyl phosphate capping enzyme (MePCE) Bmc1/Bin3 is a stable component of the S. pombe telomerase holoenzyme. Bmc1 associates with the telomerase holoenzyme and U6 snRNA through an interaction with the recently described LARP7 family member Pof8, and we demonstrate that these two factors are evolutionarily linked in fungi. Our data suggest that the association of Bmc1 with telomerase is independent of its methyltransferase activity, but rather that Bmc1 functions in telomerase holoenzyme assembly by promoting TER1 accumulation and Pof8 recruitment to TER1. Taken together, this work yields new insight into the composition, assembly, and regulation of the telomerase holoenzyme in fission yeast as well as the breadth of its evolutionary conservation.
The LARP7 complex in higher eukaryotes
Summary of findings:
Faithful segregation of sister chromatids requires the attachment of each kinetochore (Kt) to microtubules (MTs) that extend from opposite spindle poles. Merotelic Kt orientation is a Kt-MT misattachment in which a single Kt binds MTs from both spindle poles rather than just one. Genetic induction of merotelic Kt attachment during anaphase in fission yeast resulted in intra-Kt stretching followed by either correction or Kt disruption. Laser ablation of spindle MTs revealed that intra-Kt stretching and merotelic correction were dependent on MT forces. The presence of multiple merotelic chromosomes linearly antagonized the spindle elongation rate, and this phenomenon could be solved numerically using a simple force balance model. Based on the predictions of our mechanical model, we provide in vivo evidence that correction of merotelic attachment in anaphase is tension dependent and requires an Ase1/Prc1-dependent mechanism that prevents spindle collapse and thus asymmetric division and/or the appearance of the cut phenotype.
Anaphase B spindle elongation -> stretch of merotelic attachment (direct evidence) -> correction of merotelic attachment (inferred)
At the same time linear correlation with number of merotelic attachments -> slower elongation
Ase1 (weak midzone) -> collapse + active shrinkage.
The plus ends of microtubules have been speculated to regulate the actin cytoskeleton for the proper positioning of sites of cell polarization and cytokinesis. In the fission yeast Schizosaccharomyces pombe, interphase microtubules and the kelch repeat protein tea1p regulate polarized cell growth. Here, we show that tea1p is directly deposited at cell tips by microtubule plus ends. Tea1p associates in large "polarisome" complexes with bud6p and for3p, a formin that assembles actin cables. Tea1p also interacts in a separate complex with the CLIP-170 protein tip1p, a microtubule plus end-binding protein that anchors tea1p to the microtubule plus end. Localization experiments suggest that tea1p and bud6p regulate formin distribution and actin cable assembly. Although single mutants still polarize, for3Δbud6Δtea1Δ triple-mutant cells lack polarity, indicating that these proteins contribute overlapping functions in cell polarization. Thus, these experiments begin to elucidate how microtubules contribute to the proper spatial regulation of actin assembly and polarized cell growth.
Genetic interactions:
Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Delta mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.
rpb9D:
rpb9(1-74), N-term
wild type:
Partial complementation in the context of CLS, ROS and menadione sensitivity production with human and Scerev -> Already annotated
NTR: Increased sensitivity to menadione in stationary phase
NTR: normal cellular reactive oxygen species level during stationary phase
Regulation of microtubule organizing centers (MTOCs) orchestrates the reorganization of the microtubule (MT) cytoskeleton. In the fission yeast Schizosaccharomyces pombe, an equatorial MTOC (eMTOC) at the cell division site disassembles after cytokinesis, and multiple interphase MTOCs (iMTOCs) appear on the nucleus. Here, we show that, upon eMTOC disassembly, small satellites carrying MTOC components such as the gamma-tubulin complex travel in both directions along interphase MTs. We identify rsp1p, an MTOC protein required for eMTOC disassembly. In rsp1 loss-of-function mutants, the eMTOC persists and organizes an abnormal microtubule aster, while iMTOCs and satellites are greatly reduced. Conversely, rsp1p overexpression inhibits eMTOC formation. Rsp1p is a J domain protein that interacts with an hsp70. Thus, our findings suggest a model in which rsp1p is part of a chaperone-based mechanism that disassembles the eMTOC into satellites, contributing to the dynamic redistribution of MTOC components for organization of interphase microtubules.
Hello,
Just sharing the info on how to show the force-directed graphs in protege, in case you ever wanted to re-use it.
Install graphviz
.
In mac with brew brew install graphviz
, else full instructions: https://graphviz.org/download/
In protege:
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