Search Results (23)

Human papillomavirus (HPV) 16 is transported in a retrograde fashion from the cell surface to the Golgi apparatus. Prior to mitosis, the virus loses association with the Golgi and, following nuclear envelope breakdown, is found associated with the condensed mitotic chromatin. The intervening steps have not been well defined. It was previously demonstrated that the virus is transported to the mitotic chromosomes in vesicles. Here, we describe the role of the endoplasmic reticulum (ER) in the post-Golgi trafficking and the importance of the ER-generated coat protein complex II (COPII) anterograde trafficking pathway in HPV infection. HPV pseudovirus (PsV) colocalized with COPII components and silencing of this pathway inhibited HPV infection. Additionally, the inner COPII coat protein, Sec24b, could be biochemically isolated in association with HPV capsid proteins. This study provides insight into the mechanism of post-Golgi HPV trafficking. Full article

Previous data show that the knockdown of the MYBL1 gene in the MDA-MB-231 cell line leads to the downregulation of VCPIP1 gene expression. In addition, MYBL1 and VCPIP1 genes are co-expressed and dysregulated in some of the same triple negative breast cancer patient samples. We propose that the co-expression of the two genes is attributed to the MYBL1 transcription factor regulation of the VCPIP1 gene. We identify the MYBL1 transcription factor binding site upstream of the VCPIP1 start site and show that the MYBL1 protein can bind to the sequence identified in the VCPIP1 promoter region. Combined with the results from the knockdown study, these data support the ability of MYBL1 to regulate the VCPIP1 gene. The VCPIP1 gene functions as a deubiquitinating enzyme involved in DNA repair, protein positioning, and the assembly of the Golgi apparatus during mitotic signaling. The transcriptional regulation of VCPIP1 by the MYBL1 gene could implicate MYBL1 in these processes, which might contribute to tumor processes in TNBC. Although both genes are involved in cell cycle regulatory mechanisms, converging signaling mechanisms have not been identified. In a separate study, we performed sequence alignment of the MYBL1 transcript variants and identified an exon unique to the canonical variant. Probes that specifically target the unique MYBL1 exon show that the exon is overexpressed in tumor cell lines compared to non-tumor breast cells. We are classifying this unique MYBL1 exon as a tumor-associated exon. Full article

Constitutive heterochromatin represents a significant fraction of eukaryotic genomes (10% in Arabidopsis, 20% in humans, 30% in D. melanogaster, and up to 85% in certain nematodes) and shares similar genetic and molecular properties in animal and plant species. Studies conducted over the last few years on D. melanogaster and other organisms led to the discovery of several functions associated with constitutive heterochromatin. This made it possible to revise the concept that this ubiquitous genomic territory is incompatible with gene expression. The aim of this review is to focus the attention on a group of protein-coding genes resident in D. melanogaster constitutive of heterochromatin, which are implicated in different steps of cell division. Full article

The sperm flagellum is essential for the transport of the genetic material toward the oocyte and thus the transmission of the genetic information to the next generation. During the haploid phase of spermatogenesis, i.e., spermiogenesis, a morphological and molecular restructuring of the male germ cell, the round spermatid, takes place that includes the silencing and compaction of the nucleus, the formation of the acrosomal vesicle from the Golgi apparatus, the formation of the sperm tail, and, finally, the shedding of excessive cytoplasm. Sperm tail formation starts in the round spermatid stage when the pair of centrioles moves toward the posterior pole of the nucleus. The sperm tail, eventually, becomes located opposed to the acrosomal vesicle, which develops at the anterior pole of the nucleus. The centriole pair tightly attaches to the nucleus, forming a nuclear membrane indentation. An articular structure is formed around the centriole pair known as the connecting piece, situated in the neck region and linking the sperm head to the tail, also named the head-to-tail coupling apparatus or, in short, HTCA. Finally, the sperm tail grows out from the distal centriole that is now transformed into the basal body of the flagellum. However, a centriole pair is found in nearly all cells of the body. In somatic cells, it accumulates a large mass of proteins, the pericentriolar material (PCM), that together constitute the centrosome, which is the main microtubule-organizing center of the cell, essential not only for the structuring of the cytoskeleton and the overall cellular organization but also for mitotic spindle formation and chromosome segregation. However, in post-mitotic (G1 or G0) cells, the centrosome is transformed into the basal body. In this case, one of the centrioles, which is always the oldest or mother centriole, grows the axoneme of a cilium. Most cells of the body carry a single cilium known as the primary cilium that serves as an antenna sensing the cell’s environment. Besides, specialized cells develop multiple motile cilia differing in substructure from the immotile primary cilia that are essential in moving fluids or cargos over the cellular surface. Impairment of cilia formation causes numerous severe syndromes that are collectively subsumed as ciliopathies. This comparative overview serves to illustrate the molecular mechanisms of basal body formation, their similarities, and dissimilarities, in somatic versus male germ cells, by discussing the involved proteins/genes and their expression, localization, and function. The review, thus, aimed to provide a deeper knowledge of the molecular players that is essential for the expansion of clinical diagnostics and treatment of male fertility disorders. Full article

Aberrant centrosome activities in mutants of Dictyostelium discoideum result in anomalies of mitotic spindles that affect the reliability of chromosome segregation. Genetic instabilities caused by these deficiencies are tolerated in multinucleate cells, which can be produced by electric-pulse induced cell fusion as a source for aberrations in the mitotic apparatus of the mutant cells. Dual-color fluorescence labeling of the microtubule system and the chromosomes in live cells revealed the variability of spindle arrangements, of centrosome-nuclear interactions, and of chromosome segregation in the atypical mitoses observed. Full article

Background and objectives: The chromosome locus 20q11.21 is a commonly amplified locus in colorectal cancer, with a prevalence of 8% to 9%. Several candidate cancer-associated genes are transcribed from the locus. The therapeutic implications of the amplification in colorectal cancer remain unclear. Materials and Methods: Preclinical cell line models of colorectal cancer included in the Cancer Cell Line Encyclopedia (CCLE) collection were examined for the presence of amplifications in 20q11.21 genes. Correlations of the presence of 20q11.21 amplifications with gene essentialities and drug sensitivities were surveyed on salient databases for determination of therapeutic leads. Results: A significant subset of colorectal cancer cell lines in the CCLE (12 of 63 cell lines, 19%) bear amplifications of genes located at 20q11.21. Cancer-associated genes of the locus include ASXL1, DNMT3B, BCL2L1, TPX2, KIF3B and POFUT1. These genes are all amplified in the 12 cell lines, but they are variably over-expressed at the mRNA level, compared to non-amplified lines. 20q11.21 amplified cell lines are sensitive to various tyrosine kinase inhibitors and are resistant to chemotherapy drugs targeting the mitotic apparatus and microtubules. CRISPR and RNAi dependencies screening revealed, besides the β-catenin and KRAS genes, a few recurrent gene dependencies in more than one cell line, including YAP1 and JUP. Conclusions: Cell line models of colorectal cancer with 20q11.21 gene amplifications display dependencies on the presence of specific genes and resistance or sensitivity to specific drugs and drug categories. Observations from in vitro models may form the basis for clinical drug development in this subtype of colorectal cancer. Genetic lesions conferring synthetic lethality to certain drugs or categories of drugs could be discovered with this approach. Full article

Acral melanoma (AM) is a rare and aggressive subtype of melanoma affecting the palms, soles, and nail apparatus with similar incidence among different ethnicities. AM is unrelated to ultraviolet radiation and has a low mutation burden but frequent chromosomal rearrangements and gene amplifications. Next generation sequencing of 33 genes and somatic copy number variation (CNV) analysis with genome-wide single nucleotide polymorphism arrays were performed in order to molecularly characterize 48 primary AMs of Italian patients in association with clinicopathological and prognostic features. BRAF was the most commonly mutated gene, followed by NRAS and TP53, whereas TERT promoter, KIT, and ARID1A were less frequently mutated. Gains and losses were recurrently found in the 1q, 6p, 7, 8q, 20 and 22 chromosomes involving PREX2, RAC1, KMT2C, BRAF, CCND1, TERT, and AKT3 genes, and in the 6q, 9, 10, 11q and 16q chromosomes including CDKN2A, PTEN, and ADAMTS18 genes, respectively. This study confirmed the variety of gene mutations and the high load of CNV in primary AM. Some genomic alterations were associated with histologic prognostic features. BRAF mutations, found with a higher rate than previously reported, correlated with a low Breslow thickness, low mitotic count, low CNV of the AMs, and with early-stage of disease. Full article

GABARAP (γ-aminobutyric acid type A receptor-associated protein) and its paralogues GABARAPL1 and GABARAPL2 comprise a subfamily of autophagy-related Atg8 proteins. They are studied extensively regarding their roles during autophagy. Originally, however, especially GABARAPL2 was discovered to be involved in intra-Golgi transport and homotypic fusion of post-mitotic Golgi fragments. Recently, a broader function of mammalian Atg8s on membrane trafficking through interaction with various soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) was suggested. By immunostaining and microscopic analysis of the Golgi network, we demonstrate the importance of the presence of individual GABARAP-type proteins on Golgi morphology. Furthermore, triple knockout (TKO) cells lacking the whole GABARAP subfamily showed impaired Golgi-dependent vesicular trafficking as assessed by imaging of fluorescently labelled ceramide. With the Golgi apparatus being central within the secretory pathway, we sought to investigate the role of the GABARAP-type proteins for cell surface protein trafficking. By analysing the surfaceome composition of TKOs, we identified a subset of cell surface proteins with altered plasma membrane localisation. Taken together, we provide novel insights into an underrated aspect of autophagy-independent functions of the GABARAP subfamily and recommend considering the potential impact of GABARAP subfamily proteins on a plethora of processes during experimental analysis of GABARAP-deficient cells not only in the autophagic context. Full article

Adult neurogenesis is modulated by many G_i-coupled receptors but the precise mechanism remains elusive. A key step for maintaining the population of neural stem cells in the adult is asymmetric cell division (ACD), a process which entails the formation of two evolutionarily conserved protein complexes that establish the cell polarity and spindle orientation. Since ACD is extremely difficult to monitor in stratified tissues such as the vertebrate brain, we employed human neural progenitor cell lines to examine the regulation of the polarity and spindle orientation complexes during neuronal differentiation. Several components of the spindle orientation complex, but not those of the polarity complex, were upregulated upon differentiation of ENStem-A and ReNcell VM neural progenitor cells. Increased expression of nuclear mitotic apparatus (NuMA), Gα_i subunit, and activators of G protein signaling (AGS3 and LGN) coincided with the appearance of a neuronal marker (β-III tubulin) and the concomitant loss of neural progenitor cell markers (nestin and Sox-2). Co-immunoprecipitation assays demonstrated that both Gα_i3 and NuMA were associated with AGS3 in differentiated ENStem-A cells. Interestingly, AGS3 appeared to preferentially interact with Gα_i3 in ENStem-A cells, and this specificity for Gα_i3 was recapitulated in co-immunoprecipitation experiments using HEK293 cells transiently overexpressing GST-tagged AGS3 and different Gα_i subunits. Moreover, the binding of Gα_i3 to AGS3 was suppressed by GTPγS and pertussis toxin. Disruption of AGS3/Gα_i3 interaction by pertussis toxin indicates that AGS3 may recognize the same site on the Gα subunit as G protein-coupled receptors. Regulatory mechanisms controlling the formation of spindle orientation complex may provide novel means to manipulate ACD which in turn may have an impact on neurogenesis. Full article

Background and Objectives: The respiratory apparatus, generally affected by highly aggressive tumors like lung cancer and mesothelioma, is rarely affected by primary malignant melanoma. The aim of this review was to identify cases of primary malignant melanoma of the lung (PMML) published in the modern scientific literature, and to describe their main clinical, pathological and therapeutic features. Materials and Methods: A systematic search of publications in the electronic database PubMed has been performed using keywords, and the references of the selected articles were checked to identify additional missing studies. Results: Globally 52 papers reporting on 76 cases were identified. Among them there were 47 reports of a single case, three papers reporting on two cases each, and two larger case series published in 1997 and 2005 including eight and 15 cases, respectively. Conclusions: PMML was generally diagnosed in middle-aged males, without any apparent correlation with cigarette smoking. It was more frequently found in the lower lobes and the left lung. The tumors were generally pigmented, composed by epithelial and/or spindle cells with large nuclei and prominent nucleoli, nuclear atypia, and numerous mitotic figures; they commonly showed immunostaining for S-100, HMB 45 and Melan-A. Early detection and surgical resection were the main determinants of survival from this rare malignancy. Full article

The stress-inducible and senescence-associated tumor suppressor SIRT4, a member of the family of mitochondrial sirtuins (SIRT3, SIRT4, and SIRT5), regulates bioenergetics and metabolism via NAD⁺-dependent enzymatic activities. Next to the known mitochondrial location, we found that a fraction of endogenous or ectopically expressed SIRT4, but not SIRT3, is present in the cytosol and predominantly localizes to centrosomes. Confocal spinning disk microscopy revealed that SIRT4 is found during the cell cycle dynamically at centrosomes with an intensity peak in G₂ and early mitosis. Moreover, SIRT4 precipitates with microtubules and interacts with structural (α,β-tubulin, γ-tubulin, TUBGCP2, TUBGCP3) and regulatory (HDAC6) microtubule components as detected by co-immunoprecipitation and mass spectrometric analyses of the mitotic SIRT4 interactome. Overexpression of SIRT4 resulted in a pronounced decrease of acetylated α-tubulin (K40) associated with altered microtubule dynamics in mitotic cells. SIRT4 or the N-terminally truncated variant SIRT4(ΔN28), which is unable to translocate into mitochondria, delayed mitotic progression and reduced cell proliferation. This study extends the functional roles of SIRT4 beyond mitochondrial metabolism and provides the first evidence that SIRT4 acts as a novel centrosomal/microtubule-associated protein in the regulation of cell cycle progression. Thus, stress-induced SIRT4 may exert its role as tumor suppressor through mitochondrial as well as extramitochondrial functions, the latter associated with its localization at the mitotic spindle apparatus. Full article

Glioblastoma multiforme (GBM) represents approximately 60% of all brain tumors in adults. This malignancy shows a high biological and genetic heterogeneity associated with exceptional aggressiveness, leading to a poor survival of patients. This review provides a summary of the basic biology of GBM cells with emphasis on cell cycle and cytoskeletal apparatus of these cells, in particular microtubules. Their involvement in the important oncosuppressive process called mitotic catastrophe will next be discussed along with select examples of microtubule-targeting agents, which are currently explored in this respect such as benzimidazole carbamate compounds. Select microtubule-targeting agents, in particular benzimidazole carbamates, induce G₂/M cell cycle arrest and mitotic catastrophe in tumor cells including GBM, resulting in phenotypically variable cell fates such as mitotic death or mitotic slippage with subsequent cell demise or permanent arrest leading to senescence. Their effect is coupled with low toxicity in normal cells and not developed chemoresistance. Given the lack of efficient cytostatics or modern molecular target-specific compounds in the treatment of GBM, drugs inducing mitotic catastrophe might offer a new, efficient alternative to the existing clinical management of this at present incurable malignancy. Full article

The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus. Full article

Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability. Full article

Mounting experimental evidence shows that non-coding RNAs (ncRNAs) serve a wide variety of biological functions. Recent studies suggest that a part of ncRNAs are critically important for supporting the structure of subcellular architectures. Here, we summarize the current literature demonstrating the role of ncRNAs and RNA-binding proteins in regulating the assembly of mitotic apparatus, especially focusing on centrosomes, kinetochores, and mitotic spindles. Full article