PMID:24790093

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.

PROconsortium/PRoteinOntology#277
pombase/curation#3267

PMID:35536002

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of pho1 Gene Expression

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.

Intro

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:
- N-term IPP kinase: forward reaction
- C-term pyrophosphatase:reverse reaction
Isolated N-term has the activity
Fission yeast phosphate homeostasis (PHO) regulon three phosphate acquisition genes:
- pho1 (cell surface acid phosphatase)
- pho84: (phosphate membrane transporter)
- tgp1: (glycerophosphate transporter)
Reressed by flancking lncRNAs prt, prt2 and nc-tgp1
- By transcribing, they displace the Pho7 transcription factor from DNA binding sites
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)~~

Main conclusions:

N-term kinase domain (1-385):
- IP6 -> IP7 + ATP hydrolysis
- Asp1 can hydrolise ATP even without substrate (and ratio between use of substrate and simple ATP hydrolysis depends on Mg2+ concentration)
- Asp1 is 22x faster on IP7 phosphorylation than on IP6, favours that instead of ATP hydrolysis.
Full prot:
- No Ip6 to IP7 due to pyrophosphatase activity

Results

Fig. 1

GO annotation extensionresidues(1-385)
Asp1(1-385):
- ~~IP6 -> IP7~~
- ATP hydrolisis with or without IP6
~~Asp1(1-385)(D333A) -> neither activity. They do the mutation on the trunacted one (see KDa in Fig. 1C).~~ Already annotated to D333A

Fig. 3

~~The enzyme uses ATP as a nucleotide~~ Implied in the GO term

Fig. 4

Asp1(1-385) cannot phosphorylate 1-IP&, only 5-IP7. Indicated in the GO term.
Asp1(1-385) favours kinase activity vs ATPase when substrate is 5-IP7. CAnnot capture

Fig. 7

Mutations on Asp1(1-385)
eliminated IP7 kinase activity or reduced product formation to less than 5% of wild type (H204A, R223A, K260A, D321A, and D333A)
did not affect IP7 kinase activity (R285A and K341A)
modestly reduced activity vis-à-vis wild type: R123A (82% of WT), R293A (64%), N335A (45%), K43A (30%), and K224A (18%)

Fig. 8

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.
- No thiamine -> lncRNA is expressed -> suppression of tgp1 promoter expression of asp1 mutant
- Thiamine -> lncRNA not expressed -> active tgp1 promoter expresses asp1 mutants.
The assay measures the Pho1 acid phosphatase activity in cells:
- Constitutively expressing the wt N-term -> high acid phosphatase activity (pressumably pho1 gene expression is induced)
  - In the wt -> activity is 5.7 vs. 157
- This is lower in mutants to alanine, lowest in those that inactivated the enzyme in vitro
- R123A lower levels

Fig. 10

Full-length enzyme: Asp1 converted 1-IP7 to IP6 but did not modify either the 5-IP7 or IP6 substrates (Fig. 10C), thereby affirming previous findings that Asp1 is specific for hydrolysis of the phosphoanhydride bond at the 1-pyrophosphate position. Already captured in GO
Asp1-H397A pyrophosphatase mutant pyrophosphatase mutant.

Fig. 12

Constitutive expression of full-length Asp1 increased the Pho1 activity
- Higher even if the pyrophosphatase is repressed.

Summary:

strong preference of Asp1 kinase for 5-IP7 versus IP6

FYPO

NTR: abolished inositol diphosphatase activity
NTR: normal inositol hexakisphosphate kinase activity

GO

Existing annotations.

pyrophosphatase-defective asp1-H397A refs 9,10. This has to be changed:
- abolished inositol 1-diphosphate 2,3,4,5,6-pentakisphosphate 1-diphosphatase activity, in https://curation.pombase.org/pombe/curs/893c5c2ae65e3990
GO:0052846 and GO:0101012 are probably the same thing

Ask the authors:

Discuss with Val

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:

increased termination of RNA polymerase II transcription
abnormal regulation of transcription by nutrient

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?

PMID25869666

Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways

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.

Results

Figure 1

Figure S1

Delayed nuclear fusion once they have congressed
- dhc1, S1D

Figure 2

Figure 3

Figure 4

Figure 5

~~Expression of dhc1 from nmt41 during interphase -> 10% localization to SPB with and without dl1~~
~~dl1 alone when overexpressed without dhc1 ectopic expression is not recruited to the SPB.~~
~~Co-expression of both dhc1 and dl1 from nmt41 brings recruitment to 90%.~~
~~ectopic expression of dli1 during interphase increases cellular levels of dhc1 as seen by western blot~~
[ ]

FYPO

FYPO:0006129. Make the term more specific. Create an issue.

GO

dhc1 -> nuclear congression
klp2 -> nuclear congression
Dli1?
Dhc1 data could be used for mins end directed IMP for GO.
klp2 localization at plus ends during karyogamy. still conditioned by Mal3. (Fig. 3)
Klp2 recruited to plus ends by Mal3 (Fig. S3A)
Dhc1 recruited to SPB during karyogamy (89% of cells), Fig. 3B
- Yamamoto et al. 1999 has shown that during horsetail dhc1 is in the cortex.
- [ ]

Existing annotations.

GO term of karyogamy from pkl1.
- This paper shows more direct evidence that pkl1 does not change karyogamy. klp2D and dhc1D always congress, but not the double mutant. In addition pkl1D klp2D is as bad as pkl1D
- In particular, 44% of the asci from klp2D,dhc1-d1D klp2D,dhc1-d1D crosses and 52% from crosses of the triple delete strain contained more than four spores per ascus, suggesting that meiosis had proceeded in the absence of karyogamy to result in catastrophic rates of spore death (Figure 4A).
GO term of plus-end for klp2.
- In Janson's paper this is not mentioned.
update cut7 minus AND plus end reference for this one: https://www.pnas.org/doi/full/10.1073/pnas.1611581113
- Crosslinking activity should also be added. GO ticket pending
Tea2 plus-end directed direct evidence: Bieling et al. 2007 speckles in Fig. 4A.
Include this as support for the GO terms, because it's better direct evidence than the existing annotion from Troxell.

Discuss with Val

GO? for dl1 since localization is co-dependent? Strong evidence from co-expression during interphase from nmt1 promoter (Fig. 5C)

PMID: 35293864 summary

PMID: 35293864

Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

Findings

Fig. 1 and supps

Microtubule growth speed decreases with anaphase progression.
Microtubule rescues occur at midzone edges.

Fig. 2 and supps

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

Fig. 3 and supps

The decrease in microtubule growth happens when the nuclear membrane bridge wraps around the spindle at dumbbell transition.

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/

Fig. 4 and supps

Preventing the dumbbell transition using cerulenin or ark1-as3 prevents the decrease in MT growth speed at late anaphase (ark1-as3 membrane phenotype not included since this is just a consequence of a failure of chromosome segregation during anaphase A, and this is well documented). Deleting les1 and nem1 reduces the decrease in microtubule growth.

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

Fig. 5 and supps

Deleting ase1 leads to the loss of the organisation of rescues. This is because Ase1 recruits the rescue factor Cls1/Peg1 to the midzone.
This in turn leads to shorter spindles at the end of anaphase and spindle collapse in cells (this was reported before in Yamashita et al. 2005, PMID:15509653).
We also alter ase1 expression levels and find:
- Ase1 overexpression reduces spindle elongation speed (previously reported in Rincon et al. 2017)
- Ase1 knockdown increases growth speed throughout anaphase

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

TODO list:

Add annotations from this paper
Check annotations from previous papers:
- Kruger et al.
  - cdc25 and klp9OE, also cell size of klp9OE? probably not included
- Yamashita et al. 2005
  - Reduced spindle length at anaphase end and anaphase onset
  - Spindle collapse
  - What about spindle collapse severity / penetrance?
- Bratman and Chang
  - Cls1 interaction and so.
  - Annotation of peg1 to bundle formation, very indirect. Should be microtubule rescue instead.

PMID:35333350

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination.

Misc

Meiotic prophase, synaptonemal complex.
Uses Hi-C:
- Crosslink DNA
- Sequence to see which fragments interact with each other and build maps.
In human somatic cells cohesin complex is associated with kleisin Rad21 -> Link sister chromatids
In human miotic cells, Rec8 is the kleisin -> Link sister chromatids as well
Cohesin complex in somatic cells has been shown to make loops that extrude part of the genome, and bring together distant regions.
Formation of the LinEs (syneptonemal complex-like structures) requires the phosphorilated Rec11
Cohesin links can form in the absence of Rec8, but cannot form lateral elements (see cartoon)

Experiment conditions

They synchronise cells:
- Background: diploid pat1-as2 (analog sensitive kinase)
- Meiosis induced by addition of ATP analogue.
- G1 arrest nitrogen starvation

Findings

Fig. 1 and Fig. S1

In vegetative growth, they can detect clustering of centromeres in the hotspots where the centromeres match. (Rab1 orientation)
In meiosis, the telomers match instead, indicating formation of the bouquet (Fig. 1 A-B).
Substraction maps (red and blue) substract the contact map of Meiosis 0hr to see the condition-specific contacts.
Diagonal lines that go from telomer to telomer in Meiosis 3h represent interactions within the same chrosome due to bouquet formation. The line is strongest close to the telomer becauset that's the tethering point I guess?
In the bottom representation (triangle) they represent the contact of each chrosome with itself. By using this triangular coordinates and not a matrix the vertical line in the center corresponds to the point of contact of a chromosome and its opposite part. The coordinates of those points are (x,L-x) where L is the length of the chromosome and x is the position from the begining of the chromosome. Then they apply some threshold along the vertical axis and see how far the strong part of the vertical line reaches. This would indicate how closely the chromosomes are paired through their telomere. Worth mentioning that this is independent of centromere position in the chromosome.

Fig. 2

NOTE:
rec10D does not form LinEs, while rec8 forms aberrant LinEs. Why rec12?

rec10 and rec12 are not needed to form the loop.
rec8 needed for the bouquet, and it was known that it was reuquired for normal horsetail.

Row in curs table	Genotype	Phenotype	Figure
	rec8D	Decreased alignment index	Fig. 2A, B

Fig. 3 and S3

By magnifying you can see diamonds in the matrix, which are small loops. These loops overlap with known regions of binding of Rec8 (Ref. 85).
Fig. 3E shows that long range interactions, TADs disappear in meiosis, at the same time that the Rec8-dependent small loops appear.
This compacts the chromosome linearly (see the cartoons of synaptonemal complexes above).

Row in curs table	Genotype	Phenotype	Figure
	rec8D	Decreased loops	Fig. 3C
	rec10D	A bit decreased loops?	Fig. 3C

Fig. 4

It was shown that Wap1 (Pombe wpl1) affects the TAD big loops, does it also affect the small loops formed by Rec8?
wpl1D has lower chromosome pairing during meiosis (they image distance between ade8 locus -lac sequence + lac binding protein tagged with GFP- and a telomeric protein, they call the system ade8-GFP). This distance is high if there is no pairing. I guess this distance is reduced if chromosomes cannot be stretched. So if longer loops are formed, they are more loose rather than closely linked?
wpl1D also reduced torsional turning during horsetail.
The meiosis loops of wlp1D were bigger than wt (Fig. 4C).
deleting wpl1D and:
- rec10D: no LinEs but still thick structures
- bunch of recombination genes: still thick structures.
- In summary, formation of these structures does not require LinEs or recombination.

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

Fig. 5 and S5

You can express Rad21 during meiosis and this compensates for deletion of Rec8 (in which way?). Is this ectopic forced with a promoter, or is it a compensation/leak? Ref. 57, 59. This does not seem to be curated if expression is induced.
Their localization on wpl1D is different.
They want to see if they can find a mutant that keeps the cohesin activity, but still forms the loops.
For that they use a background of rad21D psc3D (mitotic cohesins) and transform with randomly mutagenised DNA of rec8. They know that colonies that live must have cohesin activity, otherwise they would die.

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

Physical interaction:
- rec8-F204S: still binds to Psm1. Co-IP (Fig. S5F).

Fig. 6 and S6

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

Fig. 7 and S7

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

Discussion

Rec8 is involved in the formation of axis-loop structure.
These structures are required for formation of the LinEs.
The LinEs are important for recombination (this was known).

Questions

diploid genotypes
Worth mentioning synchronisation in experiments?
- For background
- For the fact itself that this condition allows induction of meiosis.
Something on the localization of ectopically expressed Rad21?
Something on the localization of Rec8-GFP specifically to these structures.
Is there a GO-term for these structures?
Difference in horsetail between rec8D and mutant?
Should the other point mutations be curated? (Fig. S5H)
They also test recombination with a plasmid. Is this something worth anotating? (Fig. S7B).
They also show phosphomimetic Rec11 and fusion Rec11-Rec10 do not prevent LinE formation or recombination defects. I guess referee asked whether Rec8 could be involved in this process. Worth curating? (Fig. S7A and C).
Regarding the wlp1 phenotype. If rec8 goes to the bottom of the loops, why would the wpl1D mutant have a higher rec8 signal. It should have fewer longer loops?

curs table questions

What is the difference between intragenic and intergenic? Is this the plasmid thing?
Annotation 7 -> Should this be kept?
Annotation 12. Where does it come from
Annotation 13, 14. I had not included them originally.

TODO

Check annotations from previous papers:
- Ref. 90: hop1D and mek1D prevent formation of LinEs
- Ref. 57, 58: Partial rescue of rec8D by expression of Rad21 during meiosis.

PMID:35286199

Mitotic spindle formation in the absence of Polo kinase.

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.

Intro

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.

Results

Fig. 1

Fig. 2

Fig. 3

include mutants
alp4-D440E plo1D:
- 30% monopolar spindles
asp1-D507G plo1Δ alive

Fig. 4

ght5D
- interphase mts persist during mitosis
- resistance to TBZ
- normal recruitment of mid1 to cytokinetic ring
ght5D plo1D
- mislocalized septum
- reduced recruitment of mid1 to cytokinetic ring
- No localization of cdc7 to the spb during metaphase
- Normal recruitment of alp6 to the spidle

Fig. 5

Synthetic lethal in ght5D plo1D:
- bub1
- hhp1

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

Fig. S3

plo1D in .08 glucose
- sensitive to TBZ

FYPO

GO

Existing annotations.

Ask the authors:

removed the synth lethal interaction for triple mutants

Discuss with Val

Viability under certain conditions
[ ]

PMID:19879140

The fission yeast TACC protein Mia1p stabilizes microtubule arrays by length-independent crosslinking.

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.

Results

Fig. 1

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

Fig. 2

Figure 3

MBP-Mia1p-D151-300
- Short lived interphase microtubule overlaps
- Less Ase1 associates with overlaps
- Increased turnover of Ase1
MBP-Mia1p-D151-300 ase1D
- Aggravated loss of mt overlaps (worse than mia1 and ase1)

Fig. S1

MBP-Alp7 capture in resin + washed proteins captures commercial tubulin (likely not yeast tubulin)

Fig. S5

mia1D ase1D -> lethal
mia1D ase1OE-> partial rescue of unbundling

FYPO

GO

Molecular function

Microtubule bundler

Biological process

Cellular component

As indicated
SPB during mitosis

Existing annotations.

Include the fact that Mal3-GFP perturbs microtubule dynamics.

Ask the authors:

Discuss with Val

Nextprot: https://www.nextprot.org/entry/NX_O43663/structures

PMID:24239120

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.

Revise from intro

cut7 rescue

Results

Figure 1

Figure S1

Figure 2

Figure 3

Figure 4

Transient abrupt spindle length decrease at anaphase onset.
- klp5 ase1
- klp6 cut7-24

Discuss with Val

cut7-24-GFP is not lethal at 37C (Fig. S2A)
Discuss lagging chromosome or delayed start with Val
Worth adding normal spindle length at metaphase for single deletions?
Special phenotype of collapse half-way. Likely if it matters it will be mentioned again.
Do we add conditions (temperature) to genetic interactions?
Lagging chromosome phenotype
Are genetic interactions directional?
Canto
- Annotate multiple alles in canto at the same time?

TODO

If cut7ts mutants are kept, add the condition of high temperature

Ask Phong

Is cut7ts rescued by klp6 and dam1? or just if you switch mid-way?
- Just switch mid-way
Remove cut7ts conditions

Go terms

Create and discuss ticket in GO for spindle
Pending discussion with Val on regulation in GO

Q: Annotating that localization of a protein depends on another one

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?

Microtubule activities in GO

To be discussed at some point

Pending go annotations

Spindle elongation during anaphase B should probably be a term.
- Annotate cut7 and klp9 here.

PMID:15857958

Roles of Pdk1p, a Fission Yeast Protein Related to Phosphoinositide-dependent Protein Kinase, in the Regulation of Mitosis and Cytokinesis

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.

Results

Fig. 2

pdk1D
- 5% > 2 nuclei
- Diploidisation (back-to-back interphase nuclear architecture as)
- Delayed anaphase onset
  - More cells with short spindles
  - More cells with condensed chromosomes
  - Longer cdc13 signal, this is clear evidence (Fig. 3)
- Intranuclear astral microtubules (Fig. 2)

Fig. 3

pdk1D (background cdc25-22)
- Delay in ring assembly -> not only caused by delay in anaphase onset (see below)
- 31% kinetochores associated with mad2 vs. 20% wt (metaphase delay may be caused by kinetochore attachment problems)
pdk1D mph1D (background cdc25-22)
- anaphase delay is rescued (no checkpoint) -> genetic interaction phenotypic suppression
- delay in ring assembly (not fully rescued)

Fig. 4

Pdk1 -> Localizes to the SPB during mitosis only, lost at late anaphase. Also to the medial-cortical region weakly, even in pressence of latrunculin.

Fig. 5

pdk1D + plo1-1 and plo1-25 + pdk1D AT COLD TEMP
- can live in sorbitol but die without it
plo1-24C pdk1D AT COLD TEMP
- lives without sorbitol
- Grows slowly

Fig. 5

At normal temp

Fig. 6

Fig. 7

pdk1D
- broad localization of mid1 to the ring-> 34%
- weird localization of cdc4 to the ring
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
- only with sorbitol
- multinucleate cells at high temp

Microtubule pushing forces

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?

PMID:15837798

Pending FYPO: pombase/fypo#4164

PMID:26031557

Loss of kinesin-14 results in aneuploidy via kinesin-5-dependent microtubule protrusions leading to chromosome cut

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.

Revise from intro

Localization data from pkl1 (added to existing annots)
https://www.pombase.org/reference/PMID:17409356. Have these dhc1 annotations being reproduced? Yes, in https://www.molbiolcell.org/doi/10.1091/mbc.e07-09-0910

Results

Figure 1

pkl1D -> 85% cells with protrusions. Already covered
pkl1D -> 8.5% mini-chromosome loss

Figure 2

Partial rescue of protrusions by OE of pkl1-GFP in msd1D (52% of cells instead of 85%) Figure 2E
~~Loss of pkl1 localization to spindle poles in msd1D (Already there)~~
msd1D 8.2% mini-chromosome loss.
- msd1D and double-deletion pkl1D msd1D both showed similar rates of minichromosome loss compared with pkl1D (Fig. 1f), with msd1D at 8.2% and pkl1D msd1D at 11% (Fig. 2f)

Figure S2

OE of motor dead pkl1 (pkl1-md ref. 30) rescues almost completely protrusions (8%)
- Pkl1md-GFP localized primarily to the spindle poles and almost completely rescued the protrusion phenotype
Pkl1md-GFPOE msd1D partial rescue only (51%)
- In contrast, in pkl1D msd1D cells, Pkl1md-GFP localized primarily to the spindle and only partially rescued the protrusion phenotype
Figure out if the allele is the same as the rigor allele

Figure 3

pkl1D delayed anaphase onset
pkl1D mad2D delayed anaphase onset, same extent.
- This is important as it indicates that the delay in pkl1D is likely not due to a delay in chromosome capture, but rather spindle formation in prophase.
pkl1D 12% chromosome cut / aneuploidy
- MT buckling during prolonged contact with the cell tip cortex—its associated chromosome mass to the medial cell division site (Fig. 3c). Subsequent cytokinesis appeared to cut through the chromosome mass, resulting in aneuploidy in 12% of mitotic cells.

Figure 4

cut7D pkl1D shorter less frequent protrusions (30%)
Aneuploidy experiments (included only genetic interaction with cdc25-22):
- cdc25-22 pkl1D rescues aneuploidy
- Starvation of pkl1D worsens aneuploidy (38%)
- Surprisignly high baseline of aneuploidy

Figure S4

Pkl1D cells that have very long protrusions (1/3) have lower elongation speed during anaphase. Maybe too fringy?
cut7D msd1D shorter less frequent protrusions (27%)

Ontology edition

FYPO

Created fypo ticket.

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.

Long and long-lived: these are likely minus end protrusions (not labeled by GFP-Mal3), and therefore less dynamic.
Short and short-lived: these are likely plus end protrusions (labeled by GFP-Mal3), and therefore more dynamic.

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:

Minus end protrusions in pkl1D cells are caused by cut7 sliding the microtubules beyond the spb, since microtubule minus ends are not anchored to the spb.
Plus ends protrusions in pkl1D cells are simply microtubules that can grow beyond the SPB. This is allowed because pkl1D is not there to focus the pole, and does not require cut7D.

Existing Annotations

Create ticket in pombase/curation.
- Add cellular component pkl1 to GO:0035974 (meiotic spb). Same publication from existing annotation.
- Actually meiotic spindle pole body is missing, because the current annotation refers to the paper where they use SPB-less spindles. probably from that one we can also add meiotic spindle pole body.
Create ticket for FYPO:0004087.
- In the pkl1D gene page, revisit FYPO:0004087. This was probably a mistake, considering protrusions as astral microtubules, but should probably be FYPO:0003787 (long mitotic spindle microtubules protruding beyond spindle pole body). It is also listed as population phenotype.

GO

Go ticket

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).

Discuss with Val

When localization changes in a certain genetic background.
Need to confirm rigor allele being the same, since it was newly in a second study.
- Suppl. data is missing from the publication. I wrote to the journal
#11
Comment on special cut phenotype, currently anotated as "cut following normal mitotic chromosome condensation"
Split annotations of protrusions in long and short
Should we include aneuploidy rescues cdc25 and pkl1? Conditions to be rescued are not shown in the graph, this is important considering that the baseline of aneuploidy seems to be quite high in this background:
- non-starved pkl1 in this background is missing, should be the reference condition for both cdc25 and pkl1
Pkl1D cells that have very long protrusions (1/3) have lower elongation speed during anaphase.
pombase/curation#3237
GO ticket minus ends

Discuss regulation in GO

pombase/curation#3250 with val

Microtubule dynamics - phenotype ontologies

Introduction

Microtubules are filaments composed of $\alpha$ and $\beta$ tubulin subunits.
They can grow or shrink only at their tips.
They are polar, with two different ends, labelled plus end and minus end.
In pombe, minus ends are not dynamic, since they are capped by $\gamma$-tubulin, but plus ends undergo the process called dynamic instability (see cartoon below):
- They alternate between two phases of persistent growth (microtubule polymerisation) and persistent shrinkage (microtubule depolymerisation).
- Transition from growth to shrinkage is called a "catastrophe".
- Transition from shrinkage to growth is called "rescue".

Current ontology

There are currently the following terms referring to microtubule dynamics, with their children:

FYPO:0000901 abnormal microtubule polymerization or depolymerization during vegetative growth

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];

FYPO:0007111 abnormal microtubule depolymerization

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];

Others

FYPO:0003193 - Normal rate of MT depol
FYPO:0003225 - Normal rate of MT pol

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)

Proposed changes

Rename FYPO:0000901 to abnormal microtubule dynamics during vegetative growth. Then make four children:
- Abnormal microtubule depolymerisation rate:
  - This can already become the parent of the terms that were specific and mentioned depolymerisation rate, such as FYPO:0003194.
  - I can revisit the annotations of the others, and re-assign them.
- Abnormal microtubule polymerisation rate:
  - Same
  - Terms from my paper specific to anaphase B should come as children
- Abnormal microtubule growth event duration:
  - Since what is measured to calculate catastrophe rates is the duration of the growth events, the phenotype should be that.
  - In addition, the term "rescue rate" is problematic, since the use of "rate" kind of implies that the rate is constant, while in reality the catastrophe rate increases with microtubule age in the absence of other proteins, and even more so in the cell with the action of molecules such as kinesins-8 (klp5/klp6).
  - Terms from my paper specific to anaphase B should come as children (klp5/klp6 phenotype)
- Abnormal microtubule rescue:
  - A bit similar to the catastrophe, rescue cannot really be measured directly, but it's distribution (ase1D perturbs distribution of rescue) or total absence can be (cls1/peg1 thermosensitive mutant prevents rescue during anaphase B). peg1 overexpression causes superstable microtubules due to excess rescue, but it's hard to provide "direct evidence" or a measurement (Bratman and Chang 2007).
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.

Requested terms - MT dynamics

For the "abnormal" microtubule polym. and depolym. rates, I don't know if the default is to already add the increased/decreased children. I guess there will be annotations for both cases, and they are typically regulated by different proteins, so probably there will not be terms without annotations.

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

Requested terms - My paper

Change in synonyms

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.

PMID:28513584

Kinesin-5-independent mitotic spindle assembly requires the antiparallel microtubule crosslinker Ase1 in fission yeast

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.

Results

Figure 1

~~cut7D pkl1D shorter spindles~~
~~cut7D pkl1D delayed meta-ana transition~~
cut7D pkl1D 30% reduction elongation spindle speed anaphase B
cut7D pkl1D mad2D severe reduction of growth

Figure S1

pkl1OE makes monopolar spindles (this was known)
pkl1OE cut7OE rescues

Figure 2

Figure 3

Figure 4

Figure S4

ase1OE slower spindle anaphase B elongation
Cls1OE rescues cut7-24.(high temp)
pka1D partially rescues cut7-24 (high temp)

Figure 5

FYPO

GO

Spindle elongation during anaphase B should probably be a term.
- Annotate cut7 and klp9 here.

Existing annotations.

Ask the authors:

cut7D pkl1D cls1-36 tetrad dissection was done at normal temp?

Discuss with Val

What phenotype to put in cut7OE pkl1OE rescue of bipolarity. If the only thing they say is that bipolarity is reached. This is a rescue so it could be added only as genetic interaction, but probably more would be better.
Is the growth on plate a "cell growth assay"? Yes
What kind of evidence is tetrad dissection for lethality? Also what to write for lethality? Inviable cell? Cell growth assay.
- FYPO:0002061 FYPO:0001387

Spindle pole in GO

And annotated proteins vs SPB, such as motors etc.

PMID:25348260

Kinesin-14 and kinesin-5 antagonistically regulate microtubule nucleation by γ-TuRC in yeast and human cells

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.

Results

Figure 2

Co-IPs

Figure 3

~~cut7D pkl1Dstronger signal of spindle tubulin~~ (not quantified)
~~Mixed culture?~~
pkl1D thick spindle

Next figures

Many studies have used the double mutant pkl1D cut7D but they never reported chromosome segreagation errors.
Also cut7D pkl1D has consistently been found to have short metaphase length in multiple backgrounds and delayed anaphase onset.

FYPO

GO

Existing annotations.

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

Is localization of fragments somethign that should be curated
- Put only the mislocalized ones.
- [ ]

Ask the authors:

Discuss with Val

Is the replacement of human function curatable / of interest?
- Complementation: https://www.pombase.org/gene/SPAC1D4.12
They report normal spindles (no single traces shown). Toda and multiple publications from our lab show cut7D pkl1D has short spindles.
It would be really nice to have all the alleles that are constructs shown in a list, I think. Perhaps we could use something that people mentioned during the conference.
Revise interactions (peptide?)

PMID:35277511

The methyl phosphate capping enzyme Bmc1/Bin3 is a stable component of the fission yeast telomerase holoenzyme

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.

Intro

The LARP7 complex in higher eukaryotes
- It works with 7SK snRNP, composed of:
  - 7SKsnRNA: transcribed by polIII
  - MePCE: Methyl Phosphatase Capping Enzyme (Bmc1 in Pombe, the gene of study in the paper)
  - HEXIM1/2: nothing said on this one
- 7SK inhibits transcription by polII by sequestering P-TEFb (transcription elongation factors) in some nuclear structures. Don't really know what is the cellular context of this inhibition, perhaps DNA replication?
- LARP7 binds to 7SK at the poly-u trailer and promotes the recruitment of MePCE.
- MePCE methyl 5' capping prevents degradation of 7sk rna, but it's binding also contributes to its stabilisation.
- MePCE also adds the methyl cap to u6 RNA (a component of the spliceosome)
- Not much is known about MePCR outside drosophila and human.
- It's overexpressed in certain cancers.
Summary of findings:
- Bmc1 binds to u6 as expected
- Bmc1 is a component of telomerase
- No cap addition to TER1
- Bmc1 + Pof8 (LARP7) association promote telomerase assembly and TER1 accumulation.

Results

Fig. 5

bmc1D -> reduced TER1 levels (qRT pCR)
pof8D -> reduced TER1 levels (qRT pCR)
rrp6D -> increased TER1 levels (qRT pCR)
bmc1D rrp6D -> increased TER1 levels
pof8D rrp6D -> normal TER1 levels
bmc1D -> shorter telomeres
As expected, a strain lacking TER1 showed a complete loss of telomeric DNA, whereas deletion of pof8 and bmc1 only resulted in shorter telomeres, suggesting that while the two proteins are important for telomere maintenance, they are not strictly required like TER1

FYPO

GO

Bmc1 is required for telomerase activity in vitro
telomerase RNA binding](https://www.pombase.org/term/GO:0070034)
Bmc1 is part of the telomerase holoenzyme
Bmc1 promotes TER1 accumulation by preventing 3' decay by the exosome

Existing annotations.

RNA modifications:
- addition of a 5’ trimethylguanosine (TMG) cap by the methyl- transferase Tgs1

Gene structure

It seems that the ter1 rna has an unusual way of being processed, which uses spliceosome machinery. If I understand correctly, the processing involves cutting the transcript at the 3' site without ligation, and some further processing that produces an RNA molecule that keeps the sequence from 1-1213. Should this be annotated to the sequence in some way?

Ask the authors:

If such annotations would be required for this RNA, are there other genes that would require similar annotations?
Why did they attempt the deletion?

Discuss with Val

Do we put links to transcriptomics datasets like the one in Fig. 1A?
Interaction data, can it be directly submitted to BioGRID? Raw data.
- Loss of interactions after treatment with benzonase, which digests nucleic acids (test that they are not mediated by a nucleic acid intermediate)
Should Bmc1 be anotated to telomerase activity? I guess not, since it does not have the enzymatic activity in itself, but it's rather part of the holoenzyme. However, in GO there is
Telomeric repeat-binding factor contributes_to telomerase activity
Is the annotation to G0 ok?

Stuff

snRNPs (pronounced "snurps"), or small nuclear ribonucleoproteins, are RNA-protein complexes that combine with unmodified pre-mRNA and various other proteins to form a spliceosome, a large RNA-protein molecular complex upon which splicing of pre-mRNA occurs.

PMID:19948483

Ase1/Prc1-dependent spindle elongation corrects merotely during anaphase in fission yeast

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.

Results

Fig. 1

rad21-K1 AT THE PERMISIVE TEMP:
- 22% merotelic attachment vs none wild-type
  - ~~75% of those are resolved~~
  - ~~The 25% reamining one are splitted in two~~
  - ~~Max distance before disruption is 1.56~~

Fig. 3

rad21-K1
- decreased elongation speed? output_of the attachments since it is linearly correlated

Fig. 4

ase1D rad21-k1
- dies at semi-permissive temperature 31
- genetic interaction: synth lethal
- spindle collapse + uniform shrinkage
- cut phenotype / diploidisation

GO

Anaphase B spindle elongation -> stretch of merotelic attachment (direct evidence) -> correction of merotelic attachment (inferred)
- Mechanistic evidence through laser ablation (Fig. 2)
At the same time linear correlation with number of merotelic attachments -> slower elongation
- When the attachment is resolved -> increase in speed again.
Ase1 (weak midzone) -> collapse + active shrinkage.

PMID:15184402

Regulation of a formin complex by the microtubule plus end protein tea1p

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.

Results

Fig. 1

Tea1 localization:
- Cell tips during interphase and mitosis
- Microtubule plus ends

Fig. 2

Fig. 4

Fig. 5:

Fig. 6

Genetic interactions:

tea1 on top of for3:
- Makes cell shape worse
- synthetic growth defect
bud6 on top of for3:
- Makes cell shape worse
- synthetic growth defect

Other

tip1 interacts with tea1 (2hybrid)
tip1D:
- non-motile tea1 dots
- Location to plus ends lost.
- GO for tip1: protein localization to microtubule plus end: localizes tea1

GO:

tea1:
- establishment of cell polarity

FYPO

This ticket: pombase/fypo#4168

GO

Existing annotations.

Ask the authors:

Discuss with Val

Annotation of 2hyb interaction of fragments.
Annotating GO localization dependency?
Annotation GO
- Considering that bud6 does not report abnormal cell shapes
How to name actin cables that are weakly labelled? Thin?
Should all synthetic growth defect be made both ways?
Annotation of has_input for localisation GO terms?

PMID:15689489

Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast

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.

Results

Fig. 1

ase1D
- Longer cell at the time of division 13 um wt vs. 15 um ase1
- Bent cells
- Septum positioning defects - slightly off-centered but still perpendicular
- Slower cell population growth (they measure doubling time)
- sensitive to MBC

Fig. 2

ase1D
- 3-9 bundles instead of 3-5
- Bundles not aligned
- No overlap of MTs in bundles evidenced by linescan

Fig. 5

ase1D
- off-centered nucleus

Fig. 6

FYPO

GO

Existing annotations.

Ask the authors:

Discuss with Val

PMID:35618649

NTR: Increased sensitivity to menadione in stationary phase

add a common parent with 'sensitive to ethanol in stationary phase'

NTR: normal cellular reactive oxygen species level during stationary phase

How to indicate that a rigor motor localizes to the SPB

PMID:15068790

Rsp1p, a J domain protein required for disassembly and assembly of microtubule organizing centers during the fission yeast cell cycle.

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.

Results

Fig. 1

Fig. 2 and 3

rsp1-1
- abolished disassembly of the eMTOC 61%
- Increased recruitment of alp4-GFP to the septum (2x)
- premature assembly of the eMTOC (spindles of 6 um instead of 12 um -wild type-)
- Sometimes the previous eMTOC persisted until the next cytok. and would form the array.
rsp1D
- abolished disassembly of the eMTOC 30%

Fig. 4 and 5

rsp1 localization:
- interphase: SPB, perinuclear dots (iMTOC), along MTs
- mitosis: SPBs and eMTOCs
- [ ]

Fig. 6

rsp1D and rsp1-1
- Loss of alp4 localization to microtubule body 77% rsp1D 84% rsp1-1
alp4 localizes to iMTOCs only during interphase

Fig. 7

rsp1 OE:
- eMTOC formation inhibited 100%
- nucleus mispositioned 70%
- Decreased number of bundles to 2-1
- Alp4 absent or greatly reduced from septum at late anaphase

FYPO

Requested terms listed in Canto - pombase/fypo#4167

GO

Existing annotations.

Ask the authors:

Discuss with Val

Graphs in protege

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:

Screen.Recording.2022-05-12.at.16.16.38.mov

manulera / discussions_curation Goto Github PK

discussions_curation's People

Contributors

Stargazers

Watchers

discussions_curation's Issues

PMID:35536002

Activities and Structure-Function Analysis of Fission Yeast Inositol Pyrophosphate (IPP) Kinase-Pyrophosphatase Asp1 and Its Impact on Regulation of pho1 Gene Expression

Intro

Main conclusions:

Results

Fig. 1

Fig. 3

Fig. 4

Fig. 7

Fig. 8

Fig. 10

Fig. 12

Summary:

FYPO

GO

Existing annotations.

Ask the authors:

Discuss with Val

PMID25869666

Microtubule minus end motors kinesin-14 and dynein drive nuclear congression in parallel pathways

Results

Figure 1

Figure S1

Figure 2

Figure 3

Figure 4

Figure 5

FYPO

GO

Existing annotations.

Discuss with Val

PMID: 35293864

Findings

Fig. 1 and supps

Fig. 2 and supps

Fig. 3 and supps

Fig. 4 and supps

Fig. 5 and supps

TODO list:

PMID:35333350

Misc

Experiment conditions

Findings

Fig. 1 and Fig. S1

Fig. 2

Fig. 3 and S3

Fig. 4

Fig. 5 and S5

Fig. 6 and S6

Fig. 7 and S7

Discussion

Questions

curs table questions

TODO

PMID:35286199

Mitotic spindle formation in the absence of Polo kinase.

Intro

Results

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. S3

FYPO

GO

Existing annotations.

Ask the authors:

Discuss with Val

PMID:19879140

The fission yeast TACC protein Mia1p stabilizes microtubule arrays by length-independent crosslinking.

Results

Fig. 1

Fig. 2