Determination of rules governing spindle pole asymmetry · PDF fileDetermination of rules...
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Determination of rules governing spindle pole asymmetry in SIN signalling In Schizosaccaromyces pombe, cytokinesis initiation and progression is governed by a group of protein kinases called the Septation Initiation Network (SIN). These proteins are recruited and progressively activated on spindle pole bodies (SPB). Interestingly, although in initial mitotic phases some SIN proteins are distributed symmetrically on both SPBs, after sister chromatid separation they become enriched on only one SPB, while fading on the other. This leads to SPB asymmetry at the end of mitosis, which may be preserved after cell division. It is known that the SIN activity is regulated by nucleotide status of the signaling GTPase, spg1p, whose nucleotide status is controlled by the GAP, cdc16p-byr4p and a putative GEF, etd1p. We are combining automated image analysis and in silico modeling to determine the rules governing the transition of SIN proteins from the symmetric to asymmetric state, which correlates with activation of the SIN. We also plan to extend this analysis to mutant cells, to study the consequences of interfering with this control circuitry. Active spindle pole Inactive spindle pole Fig. 1. Distribution of SIN proteins on active and inactive SPB. (a) SIN signalling originates from the SPB where it becomes activated in early mitosis. During mitotic progression, active signalling is though to come from then new spindle pole body (nSPB). Signalling from the old SPB is silenced by reassembly of the GAP. The synergy of these two processes leads to tight temporal control of ring assembly, septum formation and cytokinesis. Mutants in which segregation is symmetrical results in deregulated cytokinesis. (b) schematic view of SIN activity of SPB during mitosis. After prophase, after SPBs separation, both SPBs recruit cdc7 protein in moderate levels. When the spindle starts to elongate status on both spindles changes. The activating cascade is turned on on new SPB and old SPB recruits inactivating the GAP. Each of these complexes is enriched on their corresponding SPBs during spindle elongation and becomes maximal at the highest length of spindle. a b n S P B Prophas e Metapha se Anaphas e A Anaphas e B PHASE 1 PHASE 2 PHASE 3 n S P B n S P B n S P B n S P B n S P B Active SPB conformation Inactive SPB conformation symmetric transition asymmetric cdc7-EGFP cnp1-mCHERRY Fig. 2. cdc7 localizes at SPBs symmetrically until spindle elongation in anaphase B. (a) The length of spindle was measured in mitotic cells for symmetric (similar intensity on both SPBs), transition (different intensity on both SPBs) and asymmetric (positive only for one SPB) states using ImageJ. (b) Using cnp1-mCHERRY protein as a reference for mitotic phase, we established mean spindle lengths for different localizations of cdc7 in mitosis. cdc7 localizes symmetrically on SPBs in phases I and II and gradually fades from oSPB during spindle elongation to become only present on nSPB at the end of phase III. a b asymmetric negative, SPB separated lagging chromosome transition byr4-EGFP cnp1-mCHERRY Fig. 4. byr4 appears on oSPB at initiation of spindle elongation. (a) The length of spindle was measured in mitotic cells for the different configurations shown on the left. byr4 appears on oSPB at the onset of spindle elongation, in anaphase B. (b) mean spindle lengths for appearance of byr4 on oSPB. Small percentage of cells showed staining for both SPBs; the reasons for this is under investigation. a b asymmetric symmetric negative, SPB separated sid1-EGFP cnp1-mCHERRY Fig. 3. sid1 appears on nSPB at initiation of spindle elongation. (a) The length of spindle was measured in mitotic cells. sid1 appears on nSPB at the onset of spindle elongation, in anaphase B, when kinetochore is attached to SPB. (b) Mean spindle lengths for appearance of sid1 on nSPB. A small percentage of cells showed symmetrical staining for sid1 in initial phases of mitosis; the reasons for this are under investigation. a b Conclusions: ! cdc7 appears after SPB separation on both SPBs. Symmetric localization correlates with short spindle ! The levels of cdc7 when symmetric on both SPBs are moderate ! After sister chromatid separation, cdc7 becomes gradually enriched on nSPB and fades from oSPB during spindle elongation ! sid1 appears on nSPB at the beginning of anaphase and its levels increase gradually with spindle elongation ! byr4 appears on oSPB and shows similar behavior to that of sid1 on nSPB ! Establishing rules in wild-type cells will help in screen for proteins that affect SIN status and cell cycle control. Future perspectives: ! Establishment of SIN kinetics in mutant genetic backgrounds ! Development of software enabling semi-automated analysis of SIN kinetics in collaboration with Prof. M. Unser ! Development of quantitative model of SIN behaviour in collaboration with Dr. I. Xenarios Paulina Wachowicz 1 , Daniel Schmitter 2 , Daniel Sage 2 , Michael Unser 2 , Lorenzo Cerutti 3 , Ioannis Xenarios 3 and Viesturs Simanis 1 1 Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 2 Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 3 Swiss-Prot Group & Vital-IT Group, Swiss Institute of Bioinformatics (SIB), Switzerland ppc89 cdc4 cdc11 SPB spg1 cdc7 sid1 cdc14 sid2 mob1 GTP spg1 cdc16 byr4 ppc89 cdc4 SPB GDP sid2 mob1
Transcript of Determination of rules governing spindle pole asymmetry · PDF fileDetermination of rules...
Determination of rules governing spindle pole asymmetry in SIN signalling
In Schizosaccaromyces pombe, cytokinesis initiation and progression is governed by a group of protein kinases called the Septation Initiation Network (SIN). These proteins are recruited and progressively activated on spindle pole bodies (SPB). Interestingly, although in initial mitotic phases some SIN proteins are distributed symmetrically on both SPBs, after sister chromatid separation they become enriched on only one SPB, while fading on the other. This leads to SPB asymmetry at the end of mitosis, which may be preserved after cell division.
It is known that the SIN activity is regulated by nucleotide status of the signaling GTPase, spg1p, whose nucleotide status is controlled by the GAP, cdc16p-byr4p and a putative GEF, etd1p. We are combining automated image analysis and in silico modeling to determine the rules governing the transition of SIN proteins from the symmetric to asymmetric state, which correlates with activation of the SIN. We also plan to extend this analysis to mutant cells, to study the consequences of interfering with this control circuitry.
Active spindle pole Inactive spindle pole
Fig. 1. Distribution of SIN proteins on active and inactive SPB. (a) SIN signalling originates from the SPB where it becomes activated in early mitosis. During mitotic progression, active signalling is though to come from then new spindle pole body (nSPB). Signalling from the old SPB is silenced by reassembly of the GAP. The synergy of these two processes leads to tight temporal control of ring assembly, septum formation and cytokinesis. Mutants in which segregation is symmetrical results in deregulated cytokinesis. (b) schematic view of SIN activity of SPB during mitosis. After prophase, after SPBs separation, both SPBs recruit cdc7 protein in moderate levels. When the spindle starts to elongate status on both spindles changes. The activating cascade is turned on on new SPB and old SPB recruits inactivating the GAP. Each of these complexes is enriched on their corresponding SPBs during spindle elongation and becomes maximal at the highest length of spindle.
a b oSPB n
SPB
Pro
phas
e M
etap
hase
A
naph
ase
A A
naph
ase
B
PHA
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1 PH
ASE
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oSPB nSPB
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Active SPB conformation Inactive SPB conformation
symmetric
transition
asymmetric
cdc7-EGFP cnp1-mCHERRY
Fig. 2. cdc7 localizes at SPBs symmetrically until spindle elongation in anaphase B. (a) The length of spindle was measured in mitotic cells for symmetric (similar intensity on both SPBs), transition (different intensity on both SPBs) and asymmetric (positive only for one SPB) states using ImageJ. (b) Using cnp1-mCHERRY protein as a reference for mitotic phase, we established mean spindle lengths for different localizations of cdc7 in mitosis. cdc7 localizes symmetrically on SPBs in phases I and II and gradually fades from oSPB during spindle elongation to become only present on nSPB at the end of phase III.
a b
asymmetric
negative, SPB separated
lagging chromosome
transition
byr4-EGFP cnp1-mCHERRY
Fig. 4. byr4 appears on oSPB at initiation of spindle elongation. (a) The length of spindle was measured in mitotic cells for the different configurations shown on the left. byr4 appears on oSPB at the onset of spindle elongation, in anaphase B. (b) mean spindle lengths for appearance of byr4 on oSPB. Small percentage of cells showed staining for both SPBs; the reasons for this is under investigation.
a b
asymmetric
symmetric
negative, SPB separated
sid1-EGFP cnp1-mCHERRY
Fig. 3. sid1 appears on nSPB at initiation of spindle elongation. (a) The length of spindle was measured in mitotic cells. sid1 appears on nSPB at the onset of spindle elongation, in anaphase B, when kinetochore is attached to SPB. (b) Mean spindle lengths for appearance of sid1 on nSPB. A small percentage of cells showed symmetrical staining for sid1 in initial phases of mitosis; the reasons for this are under investigation.
a b
Conclusions: ! cdc7 appears after SPB separation on both SPBs. Symmetric localization correlates with short spindle ! The levels of cdc7 when symmetric on both SPBs are moderate ! After sister chromatid separation, cdc7 becomes gradually enriched on nSPB and fades from oSPB during spindle elongation ! sid1 appears on nSPB at the beginning of anaphase and its levels increase gradually with spindle elongation ! byr4 appears on oSPB and shows similar behavior to that of sid1 on nSPB ! Establishing rules in wild-type cells will help in screen for proteins that affect SIN status and cell cycle control.
Future perspectives: ! Establishment of SIN kinetics in mutant genetic backgrounds ! Development of software enabling semi-automated analysis of SIN kinetics in collaboration with Prof. M. Unser ! Development of quantitative model of SIN behaviour in collaboration with Dr. I. Xenarios
Paulina Wachowicz1, Daniel Schmitter2, Daniel Sage2, Michael Unser2, Lorenzo Cerutti3, Ioannis Xenarios3 and Viesturs Simanis1 1Cell Cycle Control Laboratory, Swiss Institute for Experimental Cancer Research (ISREC), Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 2Biomedical Imaging Group, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland 3Swiss-Prot Group & Vital-IT Group, Swiss Institute of Bioinformatics (SIB), Switzerland
ppc8
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spg1 cdc7
sid1 cdc14
sid2 mob1
GTP
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spg1 cdc16 byr4
ppc8
9 cd
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SPB
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sid2 mob1
