Faculty Bibliography
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110
RZZ-Spindly and CENP-E form an integrated platform to recruit dynein to the kinetochore corona
Chromosome biorientation on the mitotic spindle is prerequisite to errorless genome inheritance. CENP-E (kinesin-7) and dynein-dynactin (DD), microtubule motors with opposite polarity, promote biorientation from the kinetochore corona, a polymeric structure whose assembly requires MPS1 kinase. The corona's building block consists of ROD, Zwilch, ZW10, and the DD adaptor Spindly (RZZS). How CENP-E and DD are scaffolded and mutually coordinated in the corona remains unclear. Here, we show that when corona assembly is prevented through MPS1 inhibition, CENP-E is absolutely required to retain RZZS at kinetochores. An RZZS phosphomimetic mutant bypasses this requirement, demonstrating the existence of a second receptor for polymeric RZZS. With active MPS1, CENP-E is dispensable for corona expansion, but strictly required for physiological kinetochore accumulation of DD. Thus, we identify the corona as an integrated scaffold where CENP-E kinesin controls DD kinetochore loading for coordinated bidirectional transport of chromosome cargo.
PMCID:10711656
PMID: 37984321
ISSN: 1460-2075
CID: 5608282
Apoptotic cell death in disease-Current understanding of the NCCD 2023
Apoptosis is a form of regulated cell death (RCD) that involves proteases of the caspase family. Pharmacological and genetic strategies that experimentally inhibit or delay apoptosis in mammalian systems have elucidated the key contribution of this process not only to (post-)embryonic development and adult tissue homeostasis, but also to the etiology of multiple human disorders. Consistent with this notion, while defects in the molecular machinery for apoptotic cell death impair organismal development and promote oncogenesis, the unwarranted activation of apoptosis promotes cell loss and tissue damage in the context of various neurological, cardiovascular, renal, hepatic, infectious, neoplastic and inflammatory conditions. Here, the Nomenclature Committee on Cell Death (NCCD) gathered to critically summarize an abundant pre-clinical literature mechanistically linking the core apoptotic apparatus to organismal homeostasis in the context of disease.
PMCID:10130819
PMID: 37100955
ISSN: 1476-5403
CID: 5465212
Allosteric regulation of CAD modulates de novo pyrimidine synthesis during the cell cycle
Metabolism is a fundamental cellular process that is coordinated with cell cycle progression. Despite this association, a mechanistic understanding of cell cycle phase-dependent metabolic pathway regulation remains elusive. Here we report the mechanism by which human de novo pyrimidine biosynthesis is allosterically regulated during the cell cycle. Combining traditional synchronization methods and metabolomics, we characterize metabolites by their accumulation pattern during cell cycle phases and identify cell cycle phase-dependent regulation of carbamoyl-phosphate synthetase 2, aspartate transcarbamylase and dihydroorotase (CAD), the first, rate-limiting enzyme in de novo pyrimidine biosynthesis. Through systematic mutational scanning and structural modelling, we find allostery as a major regulatory mechanism that controls the activity change of CAD during the cell cycle. Specifically, we report evidence of two Animalia-specific loops in the CAD allosteric domain that involve sensing and binding of uridine 5'-triphosphate, a CAD allosteric inhibitor. Based on homology with a mitochondrial carbamoyl-phosphate synthetase homologue, we identify a critical role for a signal transmission loop in regulating the formation of a substrate channel, thereby controlling CAD activity.
PMID: 36747088
ISSN: 2522-5812
CID: 5422782
KIF24 depletion induces clustering of supernumerary centrosomes in PDAC cells
Clustering of supernumerary centrosomes, which potentially leads to cell survival and chromosomal instability, is frequently observed in cancers. However, the molecular mechanisms that control centrosome clustering remain largely unknown. The centrosomal kinesin KIF24 was previously shown to restrain the assembly of primary cilia in mammalian cells. Here, we revealed that KIF24 depletion suppresses multipolar spindle formation by clustering centrosomes in pancreatic ductal adenocarcinoma (PDAC) cells harboring supernumerary centrosomes. KIF24 depletion also induced hyper-proliferation and improved mitotic progression in PDAC cells. In contrast, disruption of primary cilia failed to affect the proliferation and spindle formation in KIF24-depleted cells. These results suggest a novel role for KIF24 in suppressing centrosome clustering independent of primary ciliation in centrosome-amplified PDAC cells.
PMCID:9270500
PMID: 35803737
ISSN: 2575-1077
CID: 5268942
Regulators of tubulin polyglutamylation control nuclear shape and cilium disassembly by balancing microtubule and actin assembly
Cytoskeletal networks play an important role in regulating nuclear morphology and ciliogenesis. However, the role of microtubule (MT) post-translational modifications in nuclear shape regulation and cilium disassembly has not been explored. Here we identified a novel regulator of the tubulin polyglutamylase complex (TPGC), C11ORF49/CSTPP1, that regulates cytoskeletal organization, nuclear shape, and cilium disassembly. Mechanistically, loss of C11ORF49/CSTPP1 impacts the assembly and stability of the TPGC, which modulates long-chain polyglutamylation levels on microtubules (MTs) and thereby balances the binding of MT-associated proteins and actin nucleators. As a result, loss of TPGC leads to aberrant, enhanced assembly of MTs that penetrate the nucleus, which in turn leads to defects in nuclear shape, and disorganization of cytoplasmic actin that disrupts the YAP/TAZ pathway and cilium disassembly. Further, we showed that C11ORF49/CSTPP1-TPGC plays mechanistically distinct roles in the regulation of nuclear shape and cilium disassembly. Remarkably, disruption of C11ORF49/CSTPP1-TPGC also leads to developmental defects in vivo. Our findings point to an unanticipated nexus that links tubulin polyglutamylation with nuclear shape and ciliogenesis.
PMID: 34782749
ISSN: 1748-7838
CID: 5049022
Cilium induction triggers differentiation of glioma stem cells
Glioblastoma multiforme (GBM) possesses glioma stem cells (GSCs) that promote self-renewal, tumor propagation, and relapse. Understanding the mechanisms of GSCs self-renewal can offer targeted therapeutic interventions. However, insufficient knowledge of GSCs' fundamental biology is a significant bottleneck hindering these efforts. Here, we show that patient-derived GSCs recruit elevated levels of proteins that ensure the temporal cilium disassembly, leading to suppressed ciliogenesis. Depleting the cilia disassembly complex components is sufficient to induce ciliogenesis in a subset of GSCs via relocating platelet-derived growth factor receptor-alpha (PDGFR-α) to a newly induced cilium. Importantly, restoring ciliogenesis enabled GSCs to switch from self-renewal to differentiation. Finally, using an organoid-based glioma invasion assay and brain xenografts in mice, we establish that ciliogenesis-induced differentiation can prevent the infiltration of GSCs into the brain. Our findings illustrate a role for cilium as a molecular switch in determining GSCs' fate and suggest cilium induction as an attractive strategy to intervene in GSCs proliferation.
PMID: 34496239
ISSN: 2211-1247
CID: 5012002
MMP-2 is a novel histone H3 N-terminal protease necessary for myogenic gene activation
BACKGROUND:Selective proteolysis of the histone H3 N-terminal tail (H3NT) is frequently observed during eukaryotic development, generating a cleaved histone H3 (H3cl) product within a small, but significant, portion of the genome. Although increasing evidence supports a regulatory role for H3NT proteolysis in gene activation, the nuclear H3NT proteases and the biological significance of H3NT proteolysis remain largely unknown. RESULTS:In this study, established cell models of skeletal myogenesis were leveraged to investigate H3NT proteolysis. These cells displayed a rapid and progressive accumulation of a single H3cl product within chromatin during myoblast differentiation. Using conventional approaches, we discovered that the canonical extracellular matrix (ECM) protease, matrix metalloproteinase 2 (MMP-2), is the principal H3NT protease of myoblast differentiation that cleaves H3 between K18-Q19. Gelatin zymography demonstrated progressive increases in nuclear MMP-2 activity, concomitant with H3cl accumulation, during myoblast differentiation. RNAi-mediated depletion of MMP-2 impaired H3NT proteolysis and resulted in defective myogenic gene activation and myoblast differentiation. Supplementation of MMP-2 ECM activity in MMP-2-depleted cells was insufficient to rescue defective H3NT proteolysis and myogenic gene activation. CONCLUSIONS:This study revealed that MMP-2 is a novel H3NT protease and the principal H3NT protease of myoblast differentiation. The results indicate that myogenic signaling induces MMP-2-dependent H3NT proteolysis at early stages of myoblast differentiation. Importantly, the results support the necessity of nuclear MMP-2 H3NT protease activity, independent of MMP-2 activity in the ECM, for myogenic gene activation and proficient myoblast differentiation.
PMCID:8130154
PMID: 34001241
ISSN: 1756-8935
CID: 4894842
A non-canonical function for Centromere-associated protein-E controls centrosome integrity and orientation of cell division
Centromere-associated protein-E (CENP-E) is a kinesin motor localizing at kinetochores. Although its mitotic functions have been well studied, it has been challenging to investigate direct consequences of CENP-E removal using conventional methods because CENP-E depletion resulted in mitotic arrest. In this study, we harnessed an auxin-inducible degron system to achieve acute degradation of CENP-E. We revealed a kinetochore-independent role for CENP-E that removes pericentriolar material 1 (PCM1) from centrosomes in late S/early G2 phase. After acute loss of CENP-E, centrosomal Polo-like kinase 1 (Plk1) localization is abrogated through accumulation of PCM1, resulting in aberrant phosphorylation and destabilization of centrosomes, which triggers shortened astral microtubules and oblique cell divisions. Furthermore, we also observed centrosome and cell division defects in cells from a microcephaly patient with mutations in CENPE. Orientation of cell division is deregulated in some microcephalic patients, and our unanticipated findings provide additional insights into how microcephaly can result from centrosomal defects.
PMID: 33742057
ISSN: 2399-3642
CID: 4819762
Elongin A regulates transcription in vivo through enhanced RNA polymerase processivity
Elongin is an RNA polymerase II (RNAPII)-associated factor that has been shown to stimulate transcriptional elongation in vitro The Elongin complex is thought to be required for transcriptional induction in response to cellular stimuli and to ubiquitinate RNAPII in response to DNA damage. Yet the impact of the Elongin complex on transcription in vivo has not been well studied. Here, we performed comprehensive studies of the role of Elongin A, the largest subunit of the Elongin complex, on RNAPII transcription genome-wide. Our results suggest that Elongin A localizes to actively transcribed regions and potential enhancers, and the level of recruitment correlated with transcription levels. We also identified a large group of factors involved in transcription as Elongin A-associated factors. In addition, we found that loss of Elongin A leads to dramatically reduced levels of Ser2-phosphorylated, but not total, RNAPII, and cells depleted of Elongin A show stronger promoter RNAPII pausing, suggesting that Elongin A may be involved in the release of paused RNAPII. Our RNA-seq studies suggest that loss of Elongin A did not alter global transcription, and unlike prior in vitro studies, we did not observe a dramatic impact on RNAPII elongation rates in our cell-based nascent RNA-seq experiments upon Elongin A depletion. Taken together, our studies provide the first comprehensive analysis of the role of Elongin A in regulating transcription in vivo Our studies also revealed that unlike prior in vitro findings, depletion of Elongin A has little impact on global transcription profiles and transcription elongation in vivo.
PMCID:7948402
PMID: 33298525
ISSN: 1083-351x
CID: 4835222
Muscle progenitor specification and myogenic differentiation are associated with changes in chromatin topology
Using Hi-C, promoter-capture Hi-C (pCHi-C), and other genome-wide approaches in skeletal muscle progenitors that inducibly express a master transcription factor, Pax7, we systematically characterize at high-resolution the spatio-temporal re-organization of compartments and promoter-anchored interactions as a consequence of myogenic commitment and differentiation. We identify key promoter-enhancer interaction motifs, namely, cliques and networks, and interactions that are dependent on Pax7 binding. Remarkably, Pax7 binds to a majority of super-enhancers, and together with a cadre of interacting transcription factors, assembles feed-forward regulatory loops. During differentiation, epigenetic memory and persistent looping are maintained at a subset of Pax7 enhancers in the absence of Pax7. We also identify and functionally validate a previously uncharacterized Pax7-bound enhancer hub that regulates the essential myosin heavy chain cluster during skeletal muscle cell differentiation. Our studies lay the groundwork for understanding the role of Pax7 in orchestrating changes in the three-dimensional chromatin conformation in muscle progenitors.
PMID: 33277476
ISSN: 2041-1723
CID: 4702792
