Phase Separation in Cell Division.

Cell division requires the assembly and organization of a microtubule spindle for the proper separation of chromosomes in mitosis and meiosis. Phase separation is an emerging paradigm for understanding spatial and temporal regulation of a variety of cellular processes, including cell division. Phase-separated condensates have been recently discovered at many structures during cell division as a possible mechanism for properly localizing, organizing, and activating proteins involved in cell division. Here, we review how these condensates play roles in regulating microtubule density and organization and spindle assembly and function and in activating some of the key players in cell division. We conclude with perspectives on areas of future research for this exciting and rapidly advancing field.

The STARD9/Kif16a kinesin associates with mitotic microtubules and regulates spindle pole assembly.

During cell division, cells form the microtubule-based mitotic spindle, a highly specialized and dynamic structure that mediates proper chromosome transmission to daughter cells. Cancer cells can show perturbed mitotic spindles and an approach in cancer treatment has been to trigger cell killing by targeting microtubule dynamics or spindle assembly. To identify and characterize proteins necessary for spindle assembly, and potential antimitotic targets, we performed a proteomic and genetic analysis of 592 mitotic microtubule copurifying proteins (MMCPs). Screening for regulators that affect both mitosis and apoptosis, we report the identification and characterization of STARD9, a kinesin-3 family member, which localizes to centrosomes and stabilizes the pericentriolar material (PCM). STARD9-depleted cells have fragmented PCM, form multipolar spindles, activate the spindle assembly checkpoint (SAC), arrest in mitosis, and undergo apoptosis. Interestingly, STARD9-depletion synergizes with the chemotherapeutic agent taxol to increase mitotic death, demonstrating that STARD9 is a mitotic kinesin and a potential antimitotic target.

Human Protein-l-isoaspartate O-Methyltransferase Domain-Containing Protein 1 (PCMTD1) Associates with Cullin-RING Ligase Proteins.

The spontaneous l-isoaspartate protein modification has been observed to negatively affect protein function. However, this modification can be reversed in many proteins in reactions initiated by the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (PCMT1). It has been hypothesized that an additional mechanism exists in which l-isoaspartate-damaged proteins are recognized and proteolytically degraded. Herein, we describe the protein-l-isoaspartate O-methyltransferase domain-containing protein 1 (PCMTD1) as a putative E3 ubiquitin ligase substrate adaptor protein. The N-terminal domain of PCMTD1 contains l-isoaspartate and S-adenosylmethionine (AdoMet) binding motifs similar to those in PCMT1. This protein also has a C-terminal domain containing suppressor of cytokine signaling (SOCS) box ubiquitin ligase recruitment motifs found in substrate receptor proteins of the Cullin-RING E3 ubiquitin ligases. We demonstrate specific PCMTD1 binding to the canonical methyltransferase cofactor S-adenosylmethionine (AdoMet). Strikingly, while PCMTD1 is able to bind AdoMet, it does not demonstrate any l-isoaspartyl methyltransferase activity under the conditions tested here. However, this protein is able to associate with the Cullin-RING proteins Elongins B and C and Cul5 in vitro and in human cells. The previously uncharacterized PCMTD1 protein may therefore provide an alternate maintenance pathway for modified proteins in mammalian cells by acting as an E3 ubiquitin ligase adaptor protein.

DUSP7 regulates the activity of ERK2 to promote proper chromosome alignment during cell division.

Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Mapping Proximity Associations of Core Spindle Assembly Checkpoint Proteins.

The spindle assembly checkpoint (SAC) is critical for sensing defective microtubule-kinetochore attachments and tension across the kinetochore and functions to arrest cells in prometaphase to allow time to repair any errors before proceeding into anaphase. Dysregulation of the SAC leads to chromosome segregation errors that have been linked to human diseases like cancer. Although much has been learned about the composition of the SAC and the factors that regulate its activity, the proximity associations of core SAC components have not been explored in a systematic manner. Here, we have taken a BioID2-proximity-labeling proteomic approach to define the proximity protein environment for each of the five core SAC proteins BUB1, BUB3, BUBR1, MAD1L1, and MAD2L1 in mitotic-enriched populations of cells where the SAC is active. These five protein association maps were integrated to generate a SAC proximity protein network that contains multiple layers of information related to core SAC protein complexes, protein-protein interactions, and proximity associations. Our analysis validated many known SAC complexes and protein-protein interactions. Additionally, it uncovered new protein associations, including the ELYS-MAD1L1 interaction that we have validated, which lend insight into the functioning of core SAC proteins and highlight future areas of investigation to better understand the SAC.

Regulation of Iron Homeostasis through Parkin-Mediated Lactoferrin Ubiquitylation.

Somatic mutations that perturb Parkin ubiquitin ligase activity and the misregulation of iron homeostasis have both been linked to Parkinson's disease. Lactotransferrin (LTF) is a member of the family of transferrin iron binding proteins that regulate iron homeostasis, and increased levels of LTF and its receptor have been observed in neurodegenerative disorders like Parkinson's disease. Here, we report that Parkin binds to LTF and ubiquitylates LTF to influence iron homeostasis. Parkin-dependent ubiquitylation of LTF occurred most often on lysines (K) 182 and 649. Substitution of K182 or K649 with alanine (K182A or K649A, respectively) led to a decrease in the level of LTF ubiquitylation, and substitution at both sites led to a major decrease in the level of LTF ubiquitylation. Importantly, Parkin-mediated ubiquitylation of LTF was critical for regulating intracellular iron levels as overexpression of LTF ubiquitylation site point mutants (K649A or K182A/K649A) led to an increase in intracellular iron levels measured by ICP-MS/MS. Consistently, RNAi-mediated depletion of Parkin led to an increase in intracellular iron levels in contrast to overexpression of Parkin that led to a decrease in intracellular iron levels. Together, these results indicate that Parkin binds to and ubiquitylates LTF to regulate intracellular iron levels. These results expand our understanding of the cellular processes that are perturbed when Parkin activity is disrupted and more broadly the mechanisms that contribute to Parkinson's disease.

Dissecting the mechanisms of cell division.

Cell division is a highly regulated and carefully orchestrated process. Understanding the mechanisms that promote proper cell division is an important step toward unraveling important questions in cell biology and human health. Early studies seeking to dissect the mechanisms of cell division used classical genetics approaches to identify genes involved in mitosis and deployed biochemical approaches to isolate and identify proteins critical for cell division. These studies underscored that post-translational modifications and cyclin-kinase complexes play roles at the heart of the cell division program. Modern approaches for examining the mechanisms of cell division, including the use of high-throughput methods to study the effects of RNAi, cDNA, and chemical libraries, have evolved to encompass a larger biological and chemical space. Here, we outline some of the classical studies that established a foundation for the field and provide an overview of recent approaches that have advanced the study of cell division.

Proteomic Analysis of the Mammalian Katanin Family of Microtubule-severing Enzymes Defines Katanin p80 subunit B-like 1 (KATNBL1) as a Regulator of Mammalian Katanin Microtubule-severing.

The Katanin family of microtubule-severing enzymes is critical for remodeling microtubule-based structures that influence cell division, motility, morphogenesis and signaling. Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). The mammalian genome also encodes two additional A-like subunits (KATNAL1 and KATNAL2) and one additional B-like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule-severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan-ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule-severing activity in vitro Interestingly, during interphase, KATNBL1 is sequestered in the nucleus through an N-terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule-severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

Fatostatin Inhibits Cancer Cell Proliferation by Affecting Mitotic Microtubule Spindle Assembly and Cell Division.

The sterol regulatory element-binding protein (SREBP) transcription factors have become attractive targets for pharmacological inhibition in the treatment of metabolic diseases and cancer. SREBPs are critical for the production and metabolism of lipids and cholesterol, which are essential for cellular homeostasis and cell proliferation. Fatostatin was recently discovered as a specific inhibitor of SREBP cleavage-activating protein (SCAP), which is required for SREBP activation. Fatostatin possesses antitumor properties including the inhibition of cancer cell proliferation, invasion, and migration, and it arrests cancer cells in G2/M phase. Although Fatostatin has been viewed as an antitumor agent due to its inhibition of SREBP and its effect on lipid metabolism, we show that Fatostatin's anticancer properties can also be attributed to its inhibition of cell division. We analyzed the effect of SREBP activity inhibitors including Fatostatin, PF-429242, and Betulin on the cell cycle and determined that only Fatostatin possessed antimitotic properties. Fatostatin inhibited tubulin polymerization, arrested cells in mitosis, activated the spindle assembly checkpoint, and triggered mitotic catastrophe and reduced cell viability. Thus Fatostatin's ability to inhibit SREBP activity and cell division could prove beneficial in treating aggressive types of cancers such as glioblastomas that have elevated lipid metabolism and fast proliferation rates and often develop resistance to current anticancer therapies.

Large-scale chemical similarity networks for target profiling of compounds identified in cell-based chemical screens.

Target identification is one of the most critical steps following cell-based phenotypic chemical screens aimed at identifying compounds with potential uses in cell biology and for developing novel disease therapies. Current in silico target identification methods, including chemical similarity database searches, are limited to single or sequential ligand analysis that have limited capabilities for accurate deconvolution of a large number of compounds with diverse chemical structures. Here, we present CSNAP (Chemical Similarity Network Analysis Pulldown), a new computational target identification method that utilizes chemical similarity networks for large-scale chemotype (consensus chemical pattern) recognition and drug target profiling. Our benchmark study showed that CSNAP can achieve an overall higher accuracy (>80%) of target prediction with respect to representative chemotypes in large (>200) compound sets, in comparison to the SEA approach (60-70%). Additionally, CSNAP is capable of integrating with biological knowledge-based databases (Uniprot, GO) and high-throughput biology platforms (proteomic, genetic, etc) for system-wise drug target validation. To demonstrate the utility of the CSNAP approach, we combined CSNAP's target prediction with experimental ligand evaluation to identify the major mitotic targets of hit compounds from a cell-based chemical screen and we highlight novel compounds targeting microtubules, an important cancer therapeutic target. The CSNAP method is freely available and can be accessed from the CSNAP web server (http://services.mbi.ucla.edu/CSNAP/).

p16 Protein and gigaxonin are associated with the ubiquitination of NFκB in cisplatin-induced senescence of cancer cells.

The molecular mechanism of p16-mediated senescence in cisplatin-treated cancer cells is not fully understood. Here we show that cisplatin treatment of head and neck cancer cells results in nuclear transport of p16 leading to a molecular modification of NFκB. Chromatin immunoprecipitation assays show that this modification is associated with the inhibition of NFκB interacting with its DNA binding sequences, leading to decreased expression of NFκB-transcribed proteins. LCMS proteomic analysis of LAP-TAP-purified proteins from HeLa cells containing a tetracycline-inducible GFP-S peptide-NFκB expression system identified gigaxonin, an ubiquitin E3 ligase adaptor, as an NFκB-interacting protein. Immunoblotting and siRNA studies confirmed the NFκB-gigaxonin interaction and the dependence of this binding on p16-NFκB binding. Using gel shift assays, we have confirmed p16-NFκB and gigaxonin-NFκB interactions. Furthermore, we have observed increased NFκB ubiquitination with cisplatin treatment that is abolished in the absence of p16 and gigaxonin expression. Analysis of 103 primary tumors has shown that increased nuclear p16 expression correlates with enhanced survival of head and neck cancer patients (p < 0.0000542), indicating the importance of nuclear p16 expression in prognosis. Finally, p16 expression is associated with reduced cytokine expression and the presence of human papilloma virus in chemoradiation-sensitive basaloid tumors. However, the absence of p16 expression is associated with enhanced cytokine expression and the absence of human papilloma virus in aggressive tumors. These results clearly demonstrate that nuclear p16 and gigaxonin play an important role in chemosensitivity of head and neck cancers through ubiquitination of NFκB.

Cul3 substrate adaptor SPOP targets Nup153 for degradation.

SPOP is a Cul3 substrate adaptor responsible for degradation of many proteins related to cell growth and proliferation. Because mutation or misregulation of SPOP drives cancer progression, understanding the suite of SPOP substrates is important to understanding regulation of cell proliferation. Here, we identify Nup153, a component of the nuclear basket of the nuclear pore complex, as a novel substrate of SPOP. SPOP and Nup153 bind to each other and colocalize at the nuclear envelope and some nuclear foci in cells. The binding interaction between SPOP and Nup153 is complex and multivalent. Nup153 is ubiquitylated and degraded upon expression of SPOPWT but not its substrate binding-deficient mutant SPOPF102C. Depletion of SPOP via RNAi leads to Nup153 stabilization. Upon loss of SPOP, the nuclear envelope localization of spindle assembly checkpoint protein Mad1, which is tethered to the nuclear envelope by Nup153, is stronger. Altogether, our results demonstrate SPOP regulates Nup153 levels and expands our understanding of the role of SPOP in protein and cellular homeostasis.

The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis.

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.

The Mammalian Family of Katanin Microtubule-Severing Enzymes.

The katanin family of microtubule-severing enzymes is critical for cytoskeletal rearrangements that affect key cellular processes like division, migration, signaling, and homeostasis. In humans, aberrant expression, or dysfunction of the katanins, is linked to developmental, proliferative, and neurodegenerative disorders. Here, we review current knowledge on the mammalian family of katanins, including an overview of evolutionary conservation, functional domain organization, and the mechanisms that regulate katanin activity. We assess the function of katanins in dividing and non-dividing cells and how their dysregulation promotes impaired ciliary signaling and defects in developmental programs (corticogenesis, gametogenesis, and neurodevelopment) and contributes to neurodegeneration and cancer. We conclude with perspectives on future katanin research that will advance our understanding of this exciting and dynamic class of disease-associated enzymes.

CANVS: an easy-to-use application for the analysis and visualization of mass spectrometry-based protein-protein interaction/association data.

The elucidation of a protein's interaction/association network is important for defining its biological function. Mass spectrometry-based proteomic approaches have emerged as powerful tools for identifying protein-protein interactions (PPIs) and protein-protein associations (PPAs). However, interactome/association experiments are difficult to interpret, considering the complexity and abundance of data that are generated. Although tools have been developed to identify protein interactions/associations quantitatively, there is still a pressing need for easy-to-use tools that allow users to contextualize their results. To address this, we developed CANVS, a computational pipeline that cleans, analyzes, and visualizes mass spectrometry-based interactome/association data. CANVS is wrapped as an interactive Shiny dashboard with simple requirements, allowing users to interface easily with the pipeline, analyze complex experimental data, and create PPI/A networks. The application integrates systems biology databases such as BioGRID and CORUM to contextualize the results. Furthermore, CANVS features a Gene Ontology tool that allows users to identify relevant GO terms in their results and create visual networks with proteins associated with relevant GO terms. Overall, CANVS is an easy-to-use application that benefits all researchers, especially those who lack an established bioinformatic pipeline and are interested in studying interactome/association data.

The myosin regulatory light chain Myl5 localizes to mitotic spindle poles and is required for proper cell division.

Myosins are ATP-dependent actin-based molecular motors critical for diverse cellular processes like intracellular trafficking, cell motility, and cell invasion. During cell division, myosin MYO10 is important for proper mitotic spindle assembly, the anchoring of the spindle to the cortex, and positioning of the spindle to the cell mid-plane. However, myosins are regulated by myosin regulatory light chains (RLCs), and whether RLCs are important for cell division has remained unexplored. Here, we have determined that the previously uncharacterized myosin RLC Myl5 associates with the mitotic spindle and is required for cell division. We show that Myl5 localizes to the leading edge and filopodia during interphase and to mitotic spindle poles and spindle microtubules during early mitosis. Importantly, depletion of Myl5 led to defects in mitotic spindle assembly, chromosome congression, and chromosome segregation and to a slower transition through mitosis. Furthermore, Myl5 bound to MYO10 in vitro and co-localized with MYO10 at the spindle poles. These results suggest that Myl5 is important for cell division and that it may be performing its function through MYO10.

Phospho-regulation of mitotic spindle assembly.

The assembly of the bipolar mitotic spindle requires the careful orchestration of a myriad of enzyme activities like protein posttranslational modifications. Among these, phosphorylation has arisen as the principle mode for spatially and temporally activating the proteins involved in early mitotic spindle assembly processes. Here, we review key kinases, phosphatases, and phosphorylation events that regulate critical aspects of these processes. We highlight key phosphorylation substrates that are important for ensuring the fidelity of centriole duplication, centrosome maturation, and the establishment of the bipolar spindle. We also highlight techniques used to understand kinase-substrate relationships and to study phosphorylation events. We conclude with perspectives on the field of posttranslational modifications in early mitotic spindle assembly.

High-throughput generation of tagged stable cell lines for proteomic analysis.

We present an optimized system for rapid generation of localization and affinity purification-tagged mammalian stable cell lines that facilitates complex purification and interacting protein identification. The improved components of this method, including the flexibility of inducible expression, circumvent issues associated with toxicity, clonal selection, sample yields and time to data acquisition. We have applied this method to the study of cell-cycle regulators and novel microtubule-associated proteins.

Fostering inclusion and diversity through research, teaching, mentoring, and outreach.

I am deeply humbled and honored to receive the American Society for Cell Biology (ASCB) Prize for Excellence in Inclusivity. Thank you to the ASCB for recognizing the contributions of faculty to inclusion and diversity in STEM and the importance of this for the advancement of science. Thank you to the Howard Hughes Medical Institute (HHMI) for your generous support of inclusivity. The prize money will be used to fund outreach activities aimed at increasing inclusion in science and to create research opportunities for students from underrepresented groups in the sciences. In this essay, I share bits of my life's story that I hope will resonate with a broad audience, especially students from underrepresented groups in STEM, and that drive my passion for inclusion and diversity. I provide points of consideration for students to enhance their preparation for science careers and for faculty to improve the current landscape of inclusion and diversity in STEM.

Menin Associates With the Mitotic Spindle and Is Important for Cell Division.

Menin is the protein mutated in patients with multiple endocrine neoplasia type 1 (MEN1) syndrome and their corresponding sporadic tumor counterparts. We have found that menin functions in promoting proper cell division. Here, we show that menin localizes to the mitotic spindle poles and the mitotic spindle during early mitosis and to the intercellular bridge microtubules during cytokinesis in HeLa cells. In our study, menin depletion led to defects in spindle assembly and chromosome congression during early mitosis, lagging chromosomes during anaphase, defective cytokinesis, multinucleated interphase cells, and cell death. In addition, pharmacological inhibition of the menin-MLL1 interaction also led to similar cell division defects. These results indicate that menin and the menin-MLL1 interaction are important for proper cell division. These results highlight a function for menin in cell division and aid our understanding of how mutation and misregulation of menin promotes tumorigenesis.