Littérature scientifique sur le sujet « Spindle position checkpoint »

Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

Proper transmission of genetic information requires correct assembly and positioning of the mitotic spindle, responsible for driving each set of sister chromatids to the two daughter cells, followed by cytokinesis. In case of altered spindle orientation, the spindle position checkpoint inhibits Tem1-dependent activation of the mitotic exit network (MEN), thus delaying mitotic exit and cytokinesis until errors are corrected. We report a functional analysis of two previously uncharacterized budding yeast proteins, Dma1 and Dma2, 58% identical to each other and homologous to human Chfr and Schizosaccharomyces pombe Dma1, both of which have been previously implicated in mitotic checkpoints. We show that Dma1 and Dma2 are involved in proper spindle positioning, likely regulating septin ring deposition at the bud neck. DMA2 overexpression causes defects in septin ring disassembly at the end of mitosis and in cytokinesis. The latter defects can be rescued by either eliminating the spindle position checkpoint protein Bub2 or overproducing its target, Tem1, both leading to MEN hyperactivation. In addition, dma1Δ dma2Δ cells fail to activate the spindle position checkpoint in response to the lack of dynein, whereas ectopic expression of DMA2 prevents unscheduled mitotic exit of spindle checkpoint mutants treated with microtubule-depolymerizing drugs. Although their primary functions remain to be defined, our data suggest that Dma1 and Dma2 might be required to ensure timely MEN activation in telophase.

An increasing number of cellular activities can be regulated by reversible lysine acetylation. Targeting the enzymes responsible for such posttranslational modifications is instrumental in defining their substrates and functions in vivo. Here we show that a Saccharomyces cerevisiae lysine deacetylase, Hos3, is asymmetrically targeted to the daughter side of the bud neck and to the daughter spindle pole body (SPB). The morphogenesis checkpoint member Hsl7 recruits Hos3 to the neck region. Cells with a defect in spindle orientation trigger Hos3 to load onto both SPBs. When associated symmetrically with both SPBs, Hos3 functions as a spindle position checkpoint (SPOC) component to inhibit mitotic exit. Neck localization of Hos3 is essential for its symmetric association with SPBs in cells with misaligned spindles. Our data suggest that Hos3 facilitates cross-talk between the morphogenesis checkpoint and the SPOC as a component of the intricate monitoring of spindle orientation after mitotic entry and before commitment to mitotic exit.

In budding yeast, the spindle position checkpoint (SPC) delays mitotic exit until the mitotic spindle moves into the neck between the mother and bud. This checkpoint works by inhibiting the mitotic exit network (MEN), a signaling cascade initiated and controlled by Tem1, a small GTPase. Tem1 is regulated by a putative guanine exchange factor, Lte1, but the function and regulation of Lte1 remains poorly understood. Here, we identify novel components of the checkpoint that operate upstream of Lte1. We present genetic evidence in agreement with existing biochemical evidence for the molecular mechanism of a pathway that links microtubule-cortex interactions with Lte1 and mitotic exit. Each component of this pathway is required for the spindle position checkpoint to delay mitotic exit until the spindle is positioned correctly.

The spindle position checkpoint in Saccharomyces cerevisiae delays mitotic exit until the spindle has moved into the mother–bud neck, ensuring that each daughter cell inherits a nucleus. The small G protein Tem1p is critical in promoting mitotic exit and is concentrated at the spindle pole destined for the bud. The presumed nucleotide exchange factor for Tem1p, Lte1p, is concentrated in the bud. These findings suggested the hypothesis that movement of the spindle pole through the neck allows Tem1p to interact with Lte1p, promoting GTP loading of Tem1p and mitotic exit. However, we report that deletion of LTE1 had little effect on the timing of mitotic exit. We also examined several mutants in which some cells inappropriately exit mitosis even though the spindle is within the mother. In some of these cells, the spindle pole body did not interact with the bud or the neck before mitotic exit. Thus, some alternative mechanism must exist to coordinate mitotic exit with spindle position. In both wild-type and mutant cells, mitotic exit was preceded by loss of cytoplasmic microtubules from the neck. Thus, the spindle position checkpoint may monitor such interactions.

How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother–daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2010.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Most cells are polarized in that they are aware of spatial cues and can respond to these cues accordingly. One major aspect of cell function that is often responsive to these polarization cues is cell division. Cell division, the process of making two cells from one progenitor, requires equal distribution of the genetic material to the two progeny cells. When polarized cells divide, an additional constraint on the segregation of the genetic material is imposed, namely, cells must divide the genetic material along axes defined by polarization cues. In eukaryotes, this problem is generally solved by the positioning of the mitotic spindle according to these spatial cues. Defects in spindle positioning can lead to the generation of cells with incorrect organelle, genetic and molecular contents, fate and/or, spatial orientation. Cells have evolved feedback mechanisms that monitor defects in spindle positioning and delay the cell cycle in response to such defects. These mechanisms are best elucidated in the budding yeast, Saccharomyces cerevisiae. The protein kinase Kin4 inhibits the Mitotic Exit Network when the spindle is mis-positioned. How Kin4 is itself regulated and whether or how Kin4 responds to spindle mis-position is not known. The work presented in this thesis elucidates the regulation of Kin4. We identify a novel spindle position checkpoint component, PP2A-Rts 1, and show that it promotes checkpoint function by enabling proper Kin4 localization. We also identify domains and sequence determinants within Kin4 that control localization and function. We present a model of how the spindle position checkpoint senses spindle position and test this model for Kin4 function. We find that the generation of positive and negative mitotic exit regulatory zones allows the cell to sense and translate the spatial information of spindle position into a chemical cell cycle signal.
by Leon Y. Chan.
Ph.D.

In most eukaryotic cells, cytokinesis is driven by a contractile actomyosin ring, which forms at the site of cell division and drives furrow ingression. In metazoans and fungi cytokinesis requires also other cytoskeletal proteins called septins. Septins are evolutionarily conserved proteins that can hydrolyse GTP and form polymers that assemble into higher order structures, such as filaments and rings. They can interact both with the actin and microtubule cytoskeleton and with membranes. Besides being involved in cytokinesis, septins are involved in many other functions, such as polarized growth, vescicle trafficking, cellular morphology and the creation of diffusion barriers. In budding yeast the first step towards cytokinesis is the assembly of a rigid septin ring composed of five different septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) at the bud neck, the constriction between the mother cell and the bud where cytokinesis takes place. The septin ring, which is formed in G1 at the site of future bud emergence, expands into a broader hourglass structure as cells bud and enter S phase, and it splits into two separate rings just before cytokinesis. These structural changes are accompanied by dynamic transitions. In fact, septins are static (during the “frozen” state) throughout most of the cell cycle and dynamic (during the “fluid” state) in late G1 and prior to cytokinesis. The transition from fluid to frozen state is promoted by protein kinases, such as Gin4 and the PAK kinase Cla4, which localize at the bud neck and directly phosphorylate septins. Conversely, the Tem1 GTPase and the PP2A protein phosphatase likely regulate the reverse transition. The yeast septin ring is also involved in proper spindle positioning, which is in turn crucial for balanced chromosome partitioning. Whenever budding yeast cells experience spindle positioning defects, they undergo anaphase within the mother cell, and then hold on in telophase with elongated spindles and high levels of mitotic CDKs. This cell cycle delay is imposed by the spindle position checkpoint (SPOC), which prevents mitotic exit and cytokinesis until errors are corrected, thus avoiding the generation of anucleate and binucleate cells. The SPOC target is the Tem1 GTPase, whose active GTP-bound form promotes a signal transduction cascade called Mitotic Exit Network (MEN) that ultimately drives cells out of mitosis by leading to inactivation of mitotic CDKs. The dimeric GTPase-activating protein (GAP) Bub2/Bfa1 keeps Tem1 inhibited until the spindle is properly aligned, thus coupling mitotic exit with nuclear division. The Kin4 protein kinase is also involved in the SPOC, by keeping Bfa1 active and by regulating the dynamics of Bub2/Bfa1 at spindle poles. In addition, the Elm1 kinase and the PP2A phosphatase contribute to the SPOC at least partly through Kin4 activation. Tem1 and several downstream MEN components are found at SPBs in a cell cycle-regulated manner and are thought to promote mitotic exit from this location. The Bub2/Bfa1 complex is found predominantly on the SPB that is pulled towards the bud, while it is present on both SPBs of misaligned spindles. Conversely, the Lte1 protein, which positively regulates Tem1, is confined in the bud from the G1/S transition to telophase, when it spreads throughout the cytoplasm of both mother cell and bud. Septins participate in the SPOC by preventing the unscheduled diffusion of Lte1 into the mother cell. Our laboratory had previously implicated the two functionally redundant ubiquitin-ligases Dma1 and Dma2 in proper septin ring positioning, cytokinesis and SPOC regulation. During my PhD, I have been trying to gain insights into the molecular mechanisms by which Dma proteins regulate these processes. Lack of both Dma1 and Dma2 compromises septin ring assembly and has additive effects with the deletion of the PAK kinase CLA4. Indeed, we found that Dma proteins are essential together with Cla4 for septin ring stabilization throughout the cell cycle. Consistently, FRAP (Fluorescence Recovery After Photobleaching) analyses revealed that concomitant deletion of DMA1 and DMA2 increases septin turnover at the bud neck, thus destabilizing the septin ring. Conversely, overexpression of DMA2 stabilizes the septin ring and delays its disassembly at the end of mitosis. Genetic analyses showed that lack of both Dma proteins is lethal when combined with the deletion or mutation in septin genes or genes involved in septin ring assembly and/or stabilization, underlying the importance of these two proteins in the control of septin ring dynamics. We also showed that the role of Dma1 and Dma2 in the SPOC is not due to Lte1 spreading in the mother cell and seems to be also independent of Bub2/Bfa1. Rather, Dma1 and Dma2 appear to control localization of the Elm1 kinase at the bud neck, thus providing a mechanistic explanation for their role in both septin dynamics and SPOC. Being Dma proteins ubiquitin ligases, we hypothesised that they could target for ubiquitylation a regulator of cytokinesis. Therefore, we carried out a genetic screen for extragenic suppressors of the lethality of dma1∆ dma2∆ cla4Δ cells. We got 44 mutants that could potentially identify targets of Dma1/2. We initially focused our attention on the dominant mutations, because some of them suppressed very efficiently the cytokinesis and septin deposition defects of dma1∆ dma2∆ cla4∆ cells. Remarkably, cloning and sequencing revealed that one of the suppressors corresponds to RHO1, encoding for the yeast counterpart of metazoan RhoA. Rho1/RhoA is a conserved GTPase that is required for the assembly and the contraction of the actomyosin ring. The RHO1 mutant allele that we have isolated in our screen is novel (RHO1-D72N) and, strikingly, three additional suppressors carried exactly the same RHO1 mutation. The RHO1-D72N allele is likely hyperactive, since we could also suppress the lethality of dma1∆ dma2∆ cla4∆ cells using known RHO1 hyperactive alleles, such as RHO1-G19V, but not by dominant-negative alleles, such as rho1-D125A. Since protein kinase C (PKC) is a major target of yeast Rho1, we tested whether a hyperactive PKC1 allele (PKC1-R398P) could also suppress the lethality of the dma1∆ dma2∆ cla4∆ triple mutant and found that this was indeed the case. In addition, both RHO1-D72N and PKC1-R398P alleles were able to suppress partially the temperature-sensitivity of cdc12 mutants, which undergo septin ring disassembly at high temperature. These data strongly argue that Rho1/Pkc1 hyperactivation stabilizes the septin ring. Indeed, genetic and FRAP analyses showed that the septin ring is more stable in RHO1 and PKC1 hyperactive mutants, while lack of these proteins destabilized it. Finally we also found that the deletion of RTS1, which promotes septin ring disassembly at the end of the cell cycle, suppresses the lethality and septin ring defects of dma1Δ dma2Δ cla4Δ cells. Altogether, our data suggest that Dma1/2 might promote septin stabilization by activating Elm1, the Rho1/Pkc1 pathway and/or inhibiting PP2ARts1, directly or indirectly. Whether these proteins are direct ubiquitylation targets of Dma1 and Dma2 is an important issue to be addressed in the future.

碩士
國立臺灣大學
分子與細胞生物學研究所
104
In the yeast Saccharomyces cerevisiae, diploid cells enter meiosis and produce four haploid spores when fermentable sugar and nitrogen resources are limited. Several stress proteins are induced during sporulation, including Hsp26. Our laboratory has discovered that there is a Hsp26-dependent spindle checkpoint to monitor spindle formation or position, and to regulate spore formation in the budding yeast. To explore the mechanism of the Hsp26-dependent spindle checkpoint in monitoring spindle positioning, we studied Tem1, one of the Hsp26-interacting proteins. Tem1 is a critical component in the mitotic exit network (MEN) pathway, it plays a sensor for proper spindle positioning between the mother cell and the daughter bud. Since the TEM1 gene is essential, we could not use a knock-out method to explore the function of Tem1 in sporulation. We put the TEM1 gene under the control of the mitosis-specific CLB2 promoter to shut down TEM1 expression in meiosis. Interestingly, sporulation frequency was increased in the CLB2p-TEM1 cells. Meiotic time course studies showed that the increase in sporulation occurs at the step of spore formation, and similar result was obtained in the hsp26 cells. In addition, the response to benomyl treatment of the CLB2p-TEM1 mutant was the same to that of the hsp26 mutant. The phenotype of the hsp26 CLB2p-TEM1 double mutant indicated that Tem1 and Hsp26 might be involved in the same pathway of the spindle checkpoint for spore formation. Our results suggest that Tem1 may be involved in the Hsp26-dependent spindle checkpoint. According to the function of Tem1 in mitosis, Tem1 might act as a sensor in the checkpoint to monitor spindle position, and in turn control spore formation. We also investigated whether the Hsp26-dependent spindle checkpoint is functional in the cells undergoing only a single meiotic division. Because cells lacking Spo13 undergo a single meiotic division, we generated hsp26 deletion strain in the spo13 background and examined its effect on sporulation. However, the sporulation frequency of the hsp26 spo13 mutant is not increased, unlike that of the hsp26 mutant. In addition, the spo13 mutant did not display a decrease in sporulation after benomyl treatment or cold-shock treatment. These observations indicated that the spindle position checkpoint has no effect in the cells undergoing only a single meiotic division.

Metaplastic breast carcinoma (MpBC) is a rare and morphologically diverse group of tumors in which a variable proportion or the entire tumor is composed of nonglandular epithelium or mesenchymal cells. It is defined by the histological presence of at least two cellular types, typically epithelial and mesenchymal components. It is composed of ductal, squamous, and/or chondroid, and spindle elements, with squamous cell carcinoma being the most frequent histological subtype. MpBC represents 0.2%–5% of all breast cancers and it is very aggressive. This type of breast cancer is typically triplenegative and is therefore not targetable with hormone therapy or anti-HER2 therapies, leaving only chemotherapeutics for management. MpBCs are known for their aggressive course and poor response to chemotherapy. PDL1/PD1 expression is a predictor of the effectiveness of immune checkpoint therapy in breast cancer. Finally, there are currently no standardized treatment guidelines specifically for MpBC2. A 42-year-old female patient, lactating, who had her only pregnancy at age 40, visited a Mastology Clinic on July 16, 2019, complaining of huge left breast pain. She did not know about her family background, as she was adopted. On physical examination, she had lactating breasts and two palpable lumps of hard consistency, contiguous, and mobile in the upper outer quadrant of the left breast, measuring 3 and 2.5 cm. Mammography described dense breasts, with no other changes and breast ultrasound revealed two solid nodules, measuring 2.7 and 0.6 cm, and a simple cyst measuring 3.4 cm, all of which were contiguous in the upper outer quadrant of the left breast — BIRADS 4. A fine-needle aspiration puncture was performed in the simple cyst, with a histopathological result of poorly differentiated malignant neoplasm with pleomorphic focus, and a core-needle biopsy, with histopathological result of breast tissue infiltrated by pleomorphic malignant neoplasm. The immunohistochemical analysis showed positive for pan cytokeratin AE1/AE3 and negative for CD45, S100, myogenin, and myodio; bringing the conclusion of poorly differentiated carcinoma, suggestive of MpBC. She received neoadjuvant chemotherapy, with doxorubicin + cyclophosphamide, but had rapid local tumor progression. A new ultrasound revealed a heterogeneous and partially delimited mass, measuring 8.8×6.1 cm — BIRADS 6. The patient underwent a left total mastectomy and axillary lymph node dissection on September 23, 2019 — without breast reconstruction, and confirmed invasive metaplastic carcinoma with chondroid-type mesenchymal differentiation, measuring 7 cm, histological grade III, nuclear grade III, associated with solid and cribriform ductal carcinoma in situ, with comedonecrosis — grade III; free surgical margins, but with axillary lymph node metastasis (8/20). The immunohistochemical analysis of the surgical specimen revealed a triple-negative carcinoma: estrogen and progesterone receptors negative, and HER2 negative. The patient had a good postoperative recovery and received radiotherapy (50 Gy). Thereafter, she received adjuvant chemotherapy with capecitabine, within which she evolved with axillary, supraclavicular, and pulmonary lymph node metastasis. The PDL1 marker showed a negative result; therefore, palliative paclitaxel and bevacizumab were prescribed. The patient rapidly evolved with worsening of the lung lesions and was hospitalized on March 9, 2020, with serious dyspnea, progressing to death on March 19, 2020.