Chromosomally unstable tumor cells specifically require KIF18A for proliferation.

Chromosomal instability (CIN) is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that compromise the mitotic spindle. Thus, CIN cells may respond differently than diploid cells to treatments that target mitotic spindle regulation. Here, we test this idea by inhibiting a subset of kinesin motor proteins involved in mitotic spindle control. KIF18A is required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but is not required in near-diploid cells. Following KIF18A inhibition, CIN tumor cells exhibit mitotic delays, multipolar spindles, and increased cell death. Sensitivity to KIF18A knockdown is strongly correlated with centrosome fragmentation, which requires dynamic microtubules but does not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.

Live-Cell Assays for Cell Stress Responses Reveal New Patterns of Cell Signaling Caused by Mutations in Rhodopsin, α-Synuclein and TDP-43.

Many neurodegenerative diseases induce high levels of sustained cellular stress and alter a number of cellular processes. To examine how different mutations associated with neurodegenerative disease affect cell stress and signaling, we created live-cell assays for endoplasmic reticulum (ER)-mediated cell stress and second messenger signaling. We first examined neurodegenerative mutations associated with direct ER stress by exploring the effect of rhodopsin mutations on ER stress and Ca2+ signaling. The rhodopsin P23H mutation, the most common mutation in autosomal dominant Retinitis Pigmentosa (RP), produced increased ER stress levels compared to wild type (WT) rhodopsin. Moreover, this increase in cell stress correlated with blunted Ca2+ signaling in a stress-dependent manner. Analysis of single-cell Ca2+ signaling profiles revealed unique Ca2+ signaling responses exist in cells expressing WT or P23H rhodopsin, consistent with the idea that second messenger signaling is affected by cell stress. To explore the use of the ER-stress biosensor in neurodegenerative diseases that may not have a direct effect on ER-mediated cell stress, we examined how various mutants of α-synuclein and TDP-43 affected ER stress. Mutants of both α-synuclein and TDP-43 associated with Parkinson's disease (PD) and Amyotrophic lateral sclerosis (ALS) demonstrated increased ER stress compared to WT proteins. To examine the effect of α-synuclein and TDP-43 mutants on cellular signaling, we created a second live-cell assay to monitor changes in cAMP signaling during expression of various forms of α-synuclein and TDP-43. The increased cell stress caused by expression of the mutant proteins was accompanied by changes in phosphodiesterase activity. Both HEK293T and SH-SY5Y cells expressing these proteins displayed a shift towards increased cAMP degradation rates, likely due to increased phosphodiesterase activity. Together these data illustrate how biosensors for cellular stress and signaling can provide nuanced, new views of neurodegenerative disease processes.

Myosin motor isoforms direct specification of actomyosin function by tropomyosins.

Myosins and tropomyosins represent two cytoskeletal proteins that often work together with actin filaments in contractile and motile cellular processes. While the specialized role of tropomyosin in striated muscle myosin-II regulation is well characterized, its role in nonmuscle myosin regulation is poorly understood. We previously showed that fission yeast tropomyosin (Cdc8p) positively regulates myosin-II (Myo2p) and myosin-V (Myo52p) motors. To understand the broader implications of this regulation we examined the role of two mammalian tropomyosins (Tpm3.1cy/Tm5NM1 and Tpm4.2cy/Tm4) recently implicated in cancer cell proliferation and metastasis. Like Cdc8p, the Tpm3.1cy and Tpm4.2cy isoforms significantly enhance Myo2p and Myo52p motor activity, converting nonprocessive Myo52p molecules into processive motors that can walk along actin tracks as single molecules. In contrast to the positive regulation of Myo2p and Myo52p, Cdc8p and the mammalian tropomyosins potently inhibited skeletal muscle myosin-II, while having negligible effects on the highly processive mammalian myosin-Va. In support of a conserved role for certain tropomyosins in regulating nonmuscle actomyosin structures, Tpm3.1cy supported normal contractile ring function in fission yeast. Our work reveals that actomyosin regulation by tropomyosin is dependent on the myosin isoform, highlighting a general role for specific isoforms of tropomyosin in sorting myosin motor outputs.

A complex regulatory network coordinating cell cycles during C. elegans development is revealed by a genome-wide RNAi screen.

The development and homeostasis of multicellular animals requires precise coordination of cell division and differentiation. We performed a genome-wide RNA interference screen in Caenorhabditis elegans to reveal the components of a regulatory network that promotes developmentally programmed cell-cycle quiescence. The 107 identified genes are predicted to constitute regulatory networks that are conserved among higher animals because almost half of the genes are represented by clear human orthologs. Using a series of mutant backgrounds to assess their genetic activities, the RNA interference clones displaying similar properties were clustered to establish potential regulatory relationships within the network. This approach uncovered four distinct genetic pathways controlling cell-cycle entry during intestinal organogenesis. The enhanced phenotypes observed for animals carrying compound mutations attest to the collaboration between distinct mechanisms to ensure strict developmental regulation of cell cycles. Moreover, we characterized ubc-25, a gene encoding an E2 ubiquitin-conjugating enzyme whose human ortholog, UBE2Q2, is deregulated in several cancers. Our genetic analyses suggested that ubc-25 acts in a linear pathway with cul-1/Cul1, in parallel to pathways employing cki-1/p27 and lin-35/pRb to promote cell-cycle quiescence. Further investigation of the potential regulatory mechanism demonstrated that ubc-25 activity negatively regulates CYE-1/cyclin E protein abundance in vivo. Together, our results show that the ubc-25-mediated pathway acts within a complex network that integrates the actions of multiple molecular mechanisms to control cell cycles during development.

Fission yeast tropomyosin specifies directed transport of myosin-V along actin cables.

A hallmark of class-V myosins is their processivity--the ability to take multiple steps along actin filaments without dissociating. Our previous work suggested, however, that the fission yeast myosin-V (Myo52p) is a nonprocessive motor whose activity is enhanced by tropomyosin (Cdc8p). Here we investigate the molecular mechanism and physiological relevance of tropomyosin-mediated regulation of Myo52p transport, using a combination of in vitro and in vivo approaches. Single molecules of Myo52p, visualized by total internal reflection fluorescence microscopy, moved processively only when Cdc8p was present on actin filaments. Small ensembles of Myo52p bound to a quantum dot, mimicking the number of motors bound to physiological cargo, also required Cdc8p for continuous motion. Although a truncated form of Myo52p that lacked a cargo-binding domain failed to support function in vivo, it still underwent actin-dependent movement to polarized growth sites. This result suggests that truncated Myo52p lacking cargo, or single molecules of wild-type Myo52p with small cargoes, can undergo processive movement along actin-Cdc8p cables in vivo. Our findings outline a mechanism by which tropomyosin facilitates sorting of transport to specific actin tracks within the cell by switching on myosin processivity.

Control of Cdc14 activity coordinates cell cycle and development in Caenorhabditis elegans.

Much of our understanding of the function and regulation of the Cdc14 family of dual-specificity phosphatases originates from studies in yeasts. In these unicellular organisms Cdc14 is an important regulator of M-phase events. In contrast, the Caenorhabditis elegans homolog, cdc-14, is not necessary for mitosis, rather it is crucial for G(1)/S regulation to establish developmental cell-cycle quiescence. Despite the importance of integrating cdc-14 regulation with development, the mechanisms by which this coordination occurs are largely unknown. Here, we demonstrate that several processes conspire to focus the activity of cdc-14. First, the cdc-14 locus can produce at least six protein variants through alternative splicing. We find that a single form, CDC-14C, is the key variant acting during vulva development. Second, CDC-14C expression is limited to a subset of cells, including vulva precursors, through post-transcriptional regulation. Lastly, the CDC-14C subcellular location, and thus its potential interactions with other regulatory proteins, is regulated by nucleocytoplasmic shuttling. We find that the active export of CDC-14C from the nucleus during interphase is dependent on members of the Cyclin D and Crm1 families. We propose that these mechanisms collaborate to restrict the activity of cdc-14 as central components of an evolutionarily conserved regulatory network to coordinate cell-cycle progression with development.

A calmodulin-related light chain from fission yeast that functions with myosin-I and PI 4-kinase.

Fission yeast myosin-I (Myo1p) not only associates with calmodulin, but also employs a second light chain called Cam2p. cam2Δ cells exhibit defects in cell polarity and growth consistent with a loss of Myo1p function. Loss of Cam2p leads to a reduction in Myo1p levels at endocytic patches and a 50% drop in the rates of Myo1p-driven actin filament motility. Thus, Cam2p plays a significant role in Myo1p function. However, further studies indicated the existence of an additional Cam2p-binding partner. Cam2p was still present at cortical patches in myo1Δ cells (or in myo1-IQ2 mutants, which lack an intact Cam2p-binding motif), whereas a cam2 null (cam2Δ) suppressed cytokinesis defects of an essential light chain (ELC) mutant known to be impaired in binding to PI 4-kinase (Pik1p). Binding studies revealed that Cam2p and the ELC compete for Pik1p. Cortical localization of Cam2p in the myo1Δ background relied on its association with Pik1p, whereas overexpression studies indicated that Cam2p, in turn, contributes to Pik1p function. The fact that the Myo1p-associated defects of a cam2Δ mutant are more potent than those of a myo1-IQ2 mutant suggests that myosin light chains can contribute to actomyosin function both directly and indirectly (via phospholipid synthesis at sites of polarized growth).

Differential regulation of unconventional fission yeast myosins via the actin track.

BACKGROUND: Fission yeast possesses three unconventional myosins: Myo1p (a class I myosin that functions at endocytic actin patches) and Myo51p and Myo52p (class V myosins that function at contractile rings and actin cables, respectively). Here we used a combination of in vivo and in vitro approaches to investigate how changes in the actin track influence the motor activity and spatial regulation of these myosins. RESULTS: We optimized the isolation of Myo1p, Myo51p, and Myo52p. All three myosins exhibited robust motor activity in ATPase and actin filament gliding assays. However, decoration of actin with tropomyosin differentially regulates the activity of these motors. Tropomyosin inhibits Myo1p by blocking its ability to form productive associations with actin filaments, whereas tropomyosin increases the actin affinity and ATPase activity of Myo51p and Myo52p. The actin filament crosslinking protein fimbrin rescues Myo1p motor activity by displacing tropomyosin from actin filaments. Consistent with our in vitro findings, fimbrin and tropomyosin have opposing effects on Myo1p function at actin patches. Defects in tropomyosin function led to shorter Myo1p patch lifetimes, whereas loss of fimbrin extended Myo1p lifetimes. Furthermore, defects in tropomyosin function decreased the efficiency of Myo52p-directed motility along actin cables in the cell. CONCLUSION: Tropomyosin promotes myosin-V motility along actin cables. Accumulation of fimbrin at actin patches relieves Myo1p from tropomyosin-mediated inhibition, ensuring maximal myosin-I motor activity at these sites. Thus, spatial regulation of myosin motor function is in part controlled by specific changes in the composition of the actin track.

Transcriptional control of cell-cycle quiescence during C. elegans development.

During the development of the C. elegans reproductive system, cells that give rise to the vulva, the vulval precursor cells (VPCs), remain quiescent for two larval stages before resuming cell division in the third larval stage. We have identified several transcriptional regulators that contribute to this temporary cell-cycle arrest. Mutation of lin-1 or lin-31, two downstream targets of the Receptor Tyrosine kinase (RTK)/Ras/MAP kinase cascade that controls VPC cell fate, disrupts the temporary VPC quiescence. We found that the LIN-1/Ets and LIN-31/FoxB transcription factors promote expression of CKI-1, a member of the p27 family of cyclin-dependent kinase inhibitors (CKIs). LIN-1 and LIN-31 promote cki-1/Kip-1 transcription prior to their inhibition through RTK/Ras/MAPK activation. Another mutation identified in the screen defined the mdt-13 TRAP240 Mediator subunit. Further analysis of the multi-subunit Mediator complex revealed that a specific subset of its components act in VPC quiescence. These components substantially overlap with the CDK-8 module implicated in transcriptional repression. Taken together, strict control of cell-cycle quiescence during VPC development involves transcriptional induction of CKI-1 and transcriptional regulation through the Mediator complex. These transcriptional regulators represent potential molecular connections between development and the basic cell-cycle machinery.