A histone deacetylase 3 and mitochondrial complex I axis regulates toxic formaldehyde production.

Cells produce considerable genotoxic formaldehyde from an unknown source. We carry out a genome-wide CRISPR-Cas9 genetic screen in metabolically engineered HAP1 cells that are auxotrophic for formaldehyde to find this cellular source. We identify histone deacetylase 3 (HDAC3) as a regulator of cellular formaldehyde production. HDAC3 regulation requires deacetylase activity, and a secondary genetic screen identifies several components of mitochondrial complex I as mediators of this regulation. Metabolic profiling indicates that this unexpected mitochondrial requirement for formaldehyde detoxification is separate from energy generation. HDAC3 and complex I therefore control the abundance of a ubiquitous genotoxic metabolite.

Myeloid cell interferon secretion restricts Zika flavivirus infection of developing and malignant human neural progenitor cells.

Zika virus (ZIKV) can infect human developing brain (HDB) progenitors resulting in epidemic microcephaly, whereas analogous cellular tropism offers treatment potential for the adult brain cancer, glioblastoma (GBM). We compared productive ZIKV infection in HDB and GBM primary tissue explants that both contain SOX2+ neural progenitors. Strikingly, although the HDB proved uniformly vulnerable to ZIKV infection, GBM was more refractory, and this correlated with an innate immune expression signature. Indeed, GBM-derived CD11b+ microglia/macrophages were necessary and sufficient to protect progenitors against ZIKV infection in a non-cell autonomous manner. Using SOX2+ GBM cell lines, we found that CD11b+-conditioned medium containing type 1 interferon beta (IFNß) promoted progenitor resistance to ZIKV, whereas inhibition of JAK1/2 signaling restored productive infection. Additionally, CD11b+ conditioned medium, and IFNß treatment rendered HDB progenitor lines and explants refractory to ZIKV. These findings provide insight into neuroprotection for HDB progenitors as well as enhanced GBM oncolytic therapies.

Mitotic spindle association of TACC3 requires Aurora-A-dependent stabilization of a cryptic α-helix.

Aurora-A regulates the recruitment of TACC3 to the mitotic spindle through a phospho-dependent interaction with clathrin heavy chain (CHC). Here, we describe the structural basis of these interactions, mediated by three motifs in a disordered region of TACC3. A hydrophobic docking motif binds to a previously uncharacterized pocket on Aurora-A that is blocked in most kinases. Abrogation of the docking motif causes a delay in late mitosis, consistent with the cellular distribution of Aurora-A complexes. Phosphorylation of Ser558 engages a conformational switch in a second motif from a disordered state, needed to bind the kinase active site, into a helical conformation. The helix extends into a third, adjacent motif that is recognized by a helical-repeat region of CHC, not a recognized phospho-reader domain. This potentially widespread mechanism of phospho-recognition provides greater flexibility to tune the molecular details of the interaction than canonical recognition motifs that are dominated by phosphate binding.

A CEP215-HSET complex links centrosomes with spindle poles and drives centrosome clustering in cancer.

Numerical centrosome aberrations underlie certain developmental abnormalities and may promote cancer. A cell maintains normal centrosome numbers by coupling centrosome duplication with segregation, which is achieved through sustained association of each centrosome with a mitotic spindle pole. Although the microcephaly- and primordial dwarfism-linked centrosomal protein CEP215 has been implicated in this process, the molecular mechanism responsible remains unclear. Here, using proteomic profiling, we identify the minus end-directed microtubule motor protein HSET as a direct binding partner of CEP215. Targeted deletion of the HSET-binding domain of CEP215 in vertebrate cells causes centrosome detachment and results in HSET depletion at centrosomes, a phenotype also observed in CEP215-deficient patient-derived cells. Moreover, in cancer cells with centrosome amplification, the CEP215-HSET complex promotes the clustering of extra centrosomes into pseudo-bipolar spindles, thereby ensuring viable cell division. Therefore, stabilization of the centrosome-spindle pole interface by the CEP215-HSET complex could promote survival of cancer cells containing supernumerary centrosomes.

Transcriptional silencing of long noncoding RNA GNG12-AS1 uncouples its transcriptional and product-related functions.

Long noncoding RNAs (lncRNAs) regulate gene expression via their RNA product or through transcriptional interference, yet a strategy to differentiate these two processes is lacking. To address this, we used multiple small interfering RNAs (siRNAs) to silence GNG12-AS1, a nuclear lncRNA transcribed in an antisense orientation to the tumour-suppressor DIRAS3. Here we show that while most siRNAs silence GNG12-AS1 post-transcriptionally, siRNA complementary to exon 1 of GNG12-AS1 suppresses its transcription by recruiting Argonaute 2 and inhibiting RNA polymerase II binding. Transcriptional, but not post-transcriptional, silencing of GNG12-AS1 causes concomitant upregulation of DIRAS3, indicating a function in transcriptional interference. This change in DIRAS3 expression is sufficient to impair cell cycle progression. In addition, the reduction in GNG12-AS1 transcripts alters MET signalling and cell migration, but these are independent of DIRAS3. Thus, differential siRNA targeting of a lncRNA allows dissection of the functions related to the process and products of its transcription.

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly.

The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.

Hitting the brakes: targeting microtubule motors in cancer.

Despite the growing number of therapies that target cancer-specific pathways, cytotoxic treatments remain important clinical tools. The rationale for targeting cell proliferation by chemotherapeutic agents stems from the assumption that tumours harbour a greater fraction of actively dividing cells than normal tissues. One such group of cytotoxic drugs impair microtubule polymers, which are cytoskeletal components of cells essential for many processes including mitosis. However, in addition to their antimitotic action, these agents cause debilitating and dose-limiting neurotoxicity because of the essential functions of microtubules in neurons. To overcome this limitation, drugs against mitosis-specific targets have been developed over the past decade, albeit with variable clinical success. Here we review the key lessons learnt from antimitotic therapies with a focus on inhibitors of microtubule motor proteins. Furthermore, based on the cancer genome data, we describe a number of motor proteins with tumour type-specific alterations, which warrant further investigation in the quest for cytotoxic targets with increased cancer specificity.

Design, synthesis, and biological evaluation of an allosteric inhibitor of HSET that targets cancer cells with supernumerary centrosomes.

Centrosomes associate with spindle poles; thus, the presence of two centrosomes promotes bipolar spindle assembly in normal cells. Cancer cells often contain supernumerary centrosomes, and to avoid multipolar mitosis and cell death, these are clustered into two poles by the microtubule motor protein HSET. We report the discovery of an allosteric inhibitor of HSET, CW069, which we designed using a methodology on an interface of chemistry and biology. Using this approach, we explored millions of compounds in silico and utilized convergent syntheses. Only compound CW069 showed marked activity against HSET in vitro. The inhibitor induced multipolar mitoses only in cells containing supernumerary centrosomes. CW069 therefore constitutes a valuable tool for probing HSET function and, by reducing the growth of cells containing supernumerary centrosomes, paves the way for new cancer therapeutics.

The essential role of centrosomal NDE1 in human cerebral cortex neurogenesis.

We investigated three families whose offspring had extreme microcephaly at birth and profound mental retardation. Brain scans and postmortem data showed that affected individuals had brains less than 10% of expected size (≤10 standard deviation) and that in addition to a massive reduction in neuron production they displayed partially deficient cortical lamination (microlissencephaly). Other body systems were apparently unaffected and overall growth was normal. We found two distinct homozygous mutations of NDE1, c.83+1G>T (p.Ala29GlnfsX114) in a Turkish family and c.684_685del (p.Pro229TrpfsX85) in two families of Pakistani origin. Using patient cells, we found that c.83+1G>T led to the use of a novel splice site and to a frameshift after NDE1 exon 2. Transfection of tagged NDE1 constructs showed that the c.684_685del mutation resulted in a NDE1 that was unable to localize to the centrosome. By staining a patient-derived cell line that carried the c.83+1G>T mutation, we found that this endogeneously expressed mutated protein equally failed to localize to the centrosome. By examining human and mouse embryonic brains, we determined that NDE1 is highly expressed in neuroepithelial cells of the developing cerebral cortex, particularly at the centrosome. We show that NDE1 accumulates on the mitotic spindle of apical neural precursors in early neurogenesis. Thus, NDE1 deficiency causes both a severe failure of neurogenesis and a deficiency in cortical lamination. Our data further highlight the importance of the centrosome in multiple aspects of neurodevelopment.

INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.

The primary cilium is an antenna-like structure that protrudes from the cell surface of quiescent/differentiated cells and participates in extracellular signal processing. Here, we report that mice deficient for the lipid 5-phosphatase Inpp5e develop a multiorgan disorder associated with structural defects of the primary cilium. In ciliated mouse embryonic fibroblasts, Inpp5e is concentrated in the axoneme of the primary cilium. Inpp5e inactivation did not impair ciliary assembly but altered the stability of pre-established cilia after serum addition. Blocking phosphoinositide 3-kinase (PI3K) activity or ciliary platelet-derived growth factor receptor alpha (PDGFRalpha) restored ciliary stability. In human INPP5E, we identified a mutation affecting INPP5E ciliary localization and cilium stability in a family with MORM syndrome, a condition related to Bardet-Biedl syndrome. Together, our results show that INPP5E plays an essential role in the primary cilium by controlling ciliary growth factor and PI3K signaling and stability, and highlight the consequences of INPP5E dysfunction.

Centrosome function in cancer: guilty or innocent?

The regulation of centrosome number and function underlies bipolar mitotic spindle formation and genetic integrity. Cancer cells both in culture and in situ exhibit a wide range of centrosome abnormalities. Here, we briefly review advances in our understanding of the pathways that govern normal centrosome function and outline the potential causes and consequences of their deregulation in disease. There is ample observational but little experimental evidence to support the conventional model that centrosome dysfunction causes genomic instability and, as a result, cancer. This model has been challenged by recent studies that have uncovered evidence of a direct link between centrosome function in asymmetric cell division and tumourigenesis. Thus, it is timely to discuss the provocative idea that, in certain tissues, abnormal centrosomes drive malignant transformation not by generating genomic instability but by deregulating asymmetric cell division.

Knock-in and knock-out: the use of reverse genetics in somatic cells to dissect mitotic pathways.

Reverse genetic methods, such as homologous gene targeting, have greatly contributed to our understanding of molecular pathways in mitosis, especially in yeast. The chicken B-lymphocyte line, DT40, represents a unique example among vertebrate somatic cells where homologous gene targeting occurs at very high frequency. DT40 cells therefore provide a useful and accessible somatic genetic system for wide-ranging biochemical and cell biological assays. In this chapter, we describe the main principles of homologous gene targeting, the concept of targeting construct design and the detailed experimental protocol of how to achieve successful knockouts. We also mention methods for conditional disruption of essential genes and conclude with specific procedures for the study of mitosis in DT40 cells.

Aurora-A: the maker and breaker of spindle poles.

The gene encoding the Aurora-A protein kinase is located in the 20q13 breast cancer amplicon and is also overexpressed in colorectal, pancreatic and gastric tumours. Although Aurora-A may not be a bona fide oncoprotein in humans, it is a promising drug target in cancer therapy. Thus, it is surprising that so little is known of its role in normal cells. The primary function of Aurora-A is to promote bipolar spindle assembly, but the molecular details of this process remained obscure until recently. The discovery of several novel Aurora-A-binding proteins and substrates has implicated Aurora-A in centrosome maturation and separation, acentrosomal and centrosomal spindle assembly, kinetochore function, cytokinesis and in cell fate determination. Here we discuss recent advances in determining the early mitotic role of Aurora-A, with a strong emphasis on its function at the mitotic spindle poles.

BRCA1-independent ubiquitination of FANCD2.

Monoubiquitination of the FANCD2 protein is a key step in the Fanconi anemia (FA) tumor suppressor pathway, coinciding with this molecule's accumulation at sites of genome damage. Strong circumstantial evidence points to a requirement for the BRCA1 gene product in this step. Here, we show that the purified BRCA1/BARD1 complex, together with E1 and UbcH5a, is sufficient to reconstitute the monoubiquitination of FANCD2 in vitro. Although siRNA-mediated knockdown of BRCA1 in human cells results in defective targeting of FANCD2 to sites of DNA damage, it does not lead to a defect in FANCD2 ubiquitination. Furthermore, ablation of the RING finger domains of either BRCA1 or BARD1 in the chicken B cell line DT40 also leaves FANCD2 modification intact. Consequently, while BRCA1 affects the accumulation of FANCD2 at sites of DNA damage, BRCA1/BARD1 E3 ligase activity is not essential for the monoubiquitination of FANCD2.

The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells.

The ch-TOG/XMAP215 family of proteins bind directly to microtubules and appear to play an essential role in stabilizing spindle microtubules. These proteins stabilize microtubules mainly by influencing microtubule plus-end dynamics, yet, in vivo, they are all strongly concentrated at spindle poles, where the minus ends of the microtubules are concentrated. In Drosophila embryos, the centrosomal protein D-TACC is required to efficiently recruit ch-TOG/Msps to centrosomes. In humans, ch-TOG and the three known TACC proteins have been implicated in cancer, but their functions are unknown. Here we extensively depleted TACC3 and ch-TOG from HeLa cells using RNA interference. In TACC3-depleted cells, spindles are well organized, but microtubules are partially destabilized and ch-TOG is no longer concentrated on spindle microtubules. In ch-TOG-depleted cells, relatively robust spindles form, but the spindles are highly disorganized. Thus, in human somatic cells, ch-TOG appears to play a major role in organizing spindle poles, and a more minor role in stabilizing spindle microtubules that is, at least in part, mediated via an interaction with TACC3.

Centrosomal TACCtics.

Although the centrosome was first described over 100 years ago, we still know relatively little of the molecular mechanisms responsible for its functions. Recently, members of a novel family of centrosomal proteins have been identified in a wide variety of organisms. The transforming acidic coiled-coil-containing (TACC) proteins all appear to play important roles in cell division and cellular organisation in both embryonic and somatic systems. These closely related molecules have been implicated in microtubule stabilisation, acentrosomal spindle assembly, translational regulation, haematopoietic development and cancer progression. In this review, I summarise what we already know of this protein family and will use the TACC proteins to illustrate the many facets that centrosomes have developed during the course of evolution.

FANCE: the link between Fanconi anaemia complex assembly and activity.

The Fanconi anaemia (FA) nuclear complex (composed of the FA proteins A, C, G and F) is essential for protection against chromosome breakage. It activates the downstream protein FANCD2 by monoubiquitylation; this then forges an association with the BRCA1 protein at sites of DNA damage. Here we show that the recently identified FANCE protein is part of this nuclear complex, binding both FANCC and FANCD2. Indeed, FANCE is required for the nuclear accumulation of FANCC and provides a critical bridge between the FA complex and FANCD2. Disease-associated FANCC mutants do not bind to FANCE, cannot accumulate in the nucleus and are unable to prevent chromosome breakage.