[Show abstract][Hide abstract] ABSTRACT: The SMN complex fulfils essential functions in the assembly of snRNPs, which are key components in splicing of pre-mRNAs. Still little is known about the regulation of SMN complex activity by posttranslational modification despite its complicated phosphorylation pattern. Previously, several phosphatases had been implicated in the regulation of SMN, including the nuclear phosphatases PPM1G and PP1γ. Here, we systematically screened all human phosphatase gene products for a regulatory role on SMN. We used the accumulation of SMN in Cajal bodies of intact proliferating cells, which actively assemble snRNPs, as a read-out for unperturbed SMN complex function. Knockdown of 29 protein phosphatases interfered with SMN accumulation in Cajal bodies suggesting impaired SMN complex function, among those the catalytically inactive, non-receptor type tyrosine phosphatase PTPN23/HD-PTP. Knockdown of PTPN23 also led to changes in the phosphorylation pattern of SMN without affecting the assembly of the SMN complex. We further show interaction between SMN and PTPN23 and finally document that PTPN23, like SMN, shuttles between nucleus and cytoplasm. Our data provide the first comprehensive screen for SMN complex regulators and establish a novel regulatory function of PTPN23 in maintaining a highly phosphorylated state of SMN important for its proper function in snRNP assembly.
Molecular Biology of the Cell 11/2014; 26(2). DOI:10.1091/mbc.E14-06-1151 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Understanding the molecular basis for proper cell division requires a detailed functional analysis of microtubule (MT) associated proteins. MT associated protein 1S (MAP1S), the most ubiquitously expressed MAP1 family member, is required for accurate cell division. Using quantitative analysis of MT plus end tracking we show here that MAP1S knockdown alters MT dynamics throughout the cell cycle. Surprisingly, MAP1S downregulation results in faster growing, yet short-lived MT in all cell cycle stages and a global loss of MT acetylation. These aberrations correlate with severe defects in the final stages of cell division. In monopolar cytokinesis assays, we demonstrate that MAP1S guides MT dependent initiation of cytokinesis. Our data underline the key role of MAP1S as a global regulator of MT stability and demonstrate a novel primary function of MAP1S to regulate MT dynamics at cytokinesis onset.
[Show abstract][Hide abstract] ABSTRACT: The survival motor neuron (SMN) complex is a macromolecular machine comprising 9 core proteins: SMN, Gemin2-8 and unrip in vertebrates. It performs tasks in RNA metabolism including the cytoplasmic assembly of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). The SMN complex also localises to the nucleus, where it accumulates in Cajal Bodies (CB) and may function in transcription and/or pre-mRNA splicing.
The SMN complex is subject to extensive phosphorylation. Detailed understanding of SMN complex regulation necessitates a comprehensive analysis of these post-translational modifications. Here, we report on the first comprehensive phosphoproteome analysis of the intact human SMN complex, which identify 48 Serine/Threonine phosphosites in the complex. We find that 7 out of 9 SMN components of the intact complex are phosphoproteins and confidently place 29 phosphorylation sites, 12 of them in SMN itself.
By the generation of multi non-phosphorylatable or phosphomimetic variants of SMN, respectively, we address to which extent phosphorylation regulates SMN complex function and localisation. Both phosphomimetic and non-phosphorylatable variants assemble into intact SMN complexes and can compensate the loss of endogenous SMN in snRNP assembly at least to some extent. However, they partially or completely fail to target to nuclear Cajal bodies. Moreover, using a mutant of SMN, which cannot be phosphorylated on previously reported tyrosine residues, we provide first evidence that this PTM regulates SMN localisation and nuclear accumulation. Our data suggest complex regulatory cues mediated by phosphorylation of serine/threonine and tyrosine residues, which control the subcellular localisation of the SMN complex and its accumulation in nuclear CB.
European journal of cell biology 03/2014; 93(3). DOI:10.1016/j.ejcb.2014.01.006 · 3.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: For more than 15 years, TPX2 has been studied as a factor critical for mitosis and spindle assembly. These functions of TPX2 are attributed to its Ran-regulated microtubule-associated protein properties and to its control of the Aurora A kinase. Overexpressed in cancers, TPX2 is being established as marker for the diagnosis and prognosis of malignancies. During interphase, TPX2 resides preferentially in the nucleus where its function had remained elusive until recently. The latest finding that TPX2 plays a role in amplification of the DNA damage response, combined with the characterization of TPX2 knockout mice, open new perspectives to understand the biology of this protein. This review provides an historic overview of the discovery of TPX2 and summarizes its cytoskeletal and signaling roles with relevance to cancer therapies. Finally, the review aims to reconcile discrepancies between the experimental and pathological effects of TPX2 overexpression and advances new roles for compartmentalized TPX2.
Cellular and Molecular Life Sciences CMLS 02/2014; 71(16). DOI:10.1007/s00018-014-1582-7 · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the γ-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.
[Show abstract][Hide abstract] ABSTRACT: In differentiated human cells, primary cilia fulfill essential functions in converting mechanical or chemical stimuli into intracellular signals. Formation and maintenance of cilia require multiple functions associated with the centriole-derived basal body, from which axonemal microtubules grow, and which assembles a gate to maintain the specific ciliary proteome. Here, we characterize the function of a novel centriolar satellite protein, SSX2IP, in the assembly of primary cilia. We show that SSX2IP localizes to the basal body of primary cilia in human and murine ciliated cells. Using siRNA knockdown in human cells, we demonstrate the importance of SSX2IP for efficient recruitment of the ciliopathy-associated satellite protein Cep290, both to satellites and the basal body. Cep290 takes a central role to gate proteins to the ciliary compartment. Consistent with that, loss of SSX2IP drastically reduces entry of the BBSome, which functions to target membrane proteins to primary cilia, and interferes with efficient accumulation of the key regulator of ciliary membrane protein targeting, Rab8. Finally, we show that SSX2IP knockdown limits targeting of the ciliary membrane protein and BBSome cargo, SSTR3, and significantly reduces axoneme length. Our data establish SSX2IP as a novel targeting factor for ciliary membrane proteins cooperating with Cep290, the BBSome and Rab8.
Molecular biology of the cell 12/2013; 25(4). DOI:10.1091/mbc.E13-09-0526 · 4.47 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Faithful action of the mitotic spindle segregates duplicated chromosomes into daughter cells. Perturbations of this process result in chromosome mis-segregation, leading to chromosomal instability and cancer development. Chromosomes are not simply passengers segregated by spindle microtubules but rather play a major active role in spindle assembly. The GTP bound form of the Ran GTPase (RanGTP), produced around chromosomes, locally activates spindle assembly factors. Recent studies have uncovered that chromosomes organize mitosis beyond spindle formation. They distinctly regulate other mitotic events, such as spindle maintenance in anaphase, which is essential for chromosome segregation. Furthermore, the direct function of chromosomes is not only to produce RanGTP but, in addition, to release key mitotic regulators from chromatin. Chromatin-remodeling factors and nuclear pore complex proteins, which have established functions on chromatin in interphase, dissociate from mitotic chromatin and function in spindle assembly or maintenance. Thus, chromosomes actively organize their own segregation using chromatin-releasing mitotic regulators as well as RanGTP.
Frontiers in Oncology 12/2013; 3:308. DOI:10.3389/fonc.2013.00308
[Show abstract][Hide abstract] ABSTRACT: The adaptive immune system is involved in tumor establishment and aggressiveness. Tumors of the ovaries, an immune-privileged organ, spread via transceolomic routes and rarely to distant organs. This is contrary to tumors of non-immune privileged organs, which often disseminate hematogenously to distant organs. Epigenetics-based immune cell quantification allows direct comparison of the immune status in benign and malignant tissues and in blood. Here, we introduce the "cellular ratio of immune tolerance" (immunoCRIT) as defined by the ratio of regulatory T cells to total T lymphocytes. The immunoCRIT was analyzed on 273 benign tissue samples of colorectal, bronchial, renal and ovarian origin as well as in 808 samples from primary colorectal, bronchial, mammary and ovarian cancers. ImmunoCRIT is strongly increased in all cancerous tissues and gradually augmented strictly dependent on tumor aggressiveness. In peripheral blood of ovarian cancer patients, immunoCRIT incrementally increases from primary diagnosis to disease recurrence, at which distant metastases frequently occur. We postulate that non-pathological immunoCRIT values observed in peripheral blood of immune privileged ovarian tumor patients are sufficient to prevent hematogenous spread at primary diagnosis. Contrarily, non-immune privileged tumors establish high immunoCRIT in an immunological environment equivalent to the bloodstream and thus spread hematogenously to distant organs. In summary, our data suggest that the immunoCRIT is a powerful marker for tumor aggressiveness and disease dissemination.
Epigenetics: official journal of the DNA Methylation Society 09/2013; 8(11). DOI:10.4161/epi.26334 · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The conserved Prp19 (pre-RNA processing 19) complex is required for pre-mRNA splicing in eukaryotic nuclei. Recent RNAi screens indicated that knockdown of Prp19 complex subunits strongly delays cell proliferation. Here we show that knockdown of the smallest subunit, BCAS2/Spf27, destabilizes the entire complex and leads to specific mitotic defects in human cells. These could result from splicing failures in interphase or reflect a direct function of the complex in open mitosis. Using Xenopus extracts, in which cell cycle progression and spindle formation can be reconstituted in vitro, we tested Prp19 complex functions during a complete cell cycle and directly in open mitosis. Strikingly, immunodepletion of the complex either before or after interphase significantly reduces the number of intact spindles, and increases the percentage of spindles with lower microtubule density and impaired metaphase alignment of chromosomes. Our data identify the Prp19 complex as the first spliceosome subcomplex that directly contributes to mitosis in vertebrates independently of its function in interphase.
PLoS ONE 09/2013; 8(9):e74851. DOI:10.1371/journal.pone.0074851 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Meiotic maturation in vertebrate oocytes is an excellent model system for microtubule reorganization during M-phase spindle assembly. Here, we surveyed changes in the pattern of microtubule-interacting proteins upon Xenopus laevis oocyte maturation by quantitative proteomics. We identified the synovial sarcoma X breakpoint protein (SSX2IP) as a novel spindle protein. Using X. laevis egg extracts, we show that SSX2IP accumulated at spindle poles in a Dynein-dependent manner and interacted with the γ-tubulin ring complex (γ-TuRC) and the centriolar satellite protein PCM-1. Immunodepletion of SSX2IP impeded γ-TuRC loading onto centrosomes. This led to reduced microtubule nucleation and spindle assembly failure. In rapidly dividing blastomeres of medaka (Oryzias latipes) and in somatic cells, SSX2IP knockdown caused fragmentation of pericentriolar material and chromosome segregation errors. We characterize SSX2IP as a novel centrosome maturation and maintenance factor that is expressed at the onset of vertebrate development. It preserves centrosome integrity and faithful mitosis during the rapid cleavage division of blastomeres and in somatic cells.
The Journal of Cell Biology 07/2013; 202(1). DOI:10.1083/jcb.201302122 · 9.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The microtubule-associated protein TPX2 plays a key role in spindle assembly and is required for mitosis in human cells. In interphase, TPX2 is actively imported into the nucleus to prevent its premature activity in microtubule organization. To date, no function has been assigned to nuclear TPX2. We now report that TPX2 plays a role in the cellular response to DNA double strand breaks induced by ionizing radiation. Loss of TPX2 leads to inordinately strong and transient accumulation of ionizing radiation-dependent Ser139-phosphorylated Histone 2AX (γ-H2AX) at G0 and G1 phases of the cell cycle. This is accompanied by the formation of increased numbers of high intensity γ-H2AX ionizing radiation-induced foci. Conversely, cells overexpressing TPX2 have reduced levels of γ-H2AX after ionizing radiation. Consistent with a role for TPX2 in the DNA damage response, we found that the protein accumulates at DNA double strand breaks and associates with the Mediator of DNA damage Checkpoint 1 (MDC1) and the Ataxia Telangiectasia Mutated (ATM) kinase, both key regulators of γ-H2AX amplification. Pharmacologic inhibition or depletion of ATM or MDC1, but not of DNA-dependent protein kinase (DNA-PK), antagonizes the γ-H2AX phenotype caused by TPX2 depletion. Importantly, the regulation of γ-H2AX signals by TPX2 is not associated with apoptosis or the mitotic functions of TPX2. In sum, our study identifies a novel and the first nuclear function for TPX2 in the cellular responses to DNA damage.
[Show abstract][Hide abstract] ABSTRACT: The mammalian DREAM complex is a key regulator of cell-cycle-regulated gene transcription and drives the expression of many gene products required for mitosis and cytokinesis. In this study, we characterized GAS2L3, which belongs to the GAS2 family of proteins with putative actin- and microtubule-binding domains as a target gene of DREAM. We found that GAS2L3 localizes to the spindle midzone and the midbody during anaphase and cytokinesis, respectively. Biochemical studies show that GAS2L3 binds to and bundles microtubules as well as F-actin in vitro. Strikingly, the RNAi-mediated knockdown of GAS2L3 results in chromosome segregation defects in multinucleated cells and in cells with multi-lobed nuclei. Likewise, chronic downregulation of GAS2L3 causes chromosome loss and aneuploidy. Time-lapse videomicroscopy experiments in GAS2L3-knockdown cells reveal abnormal oscillation of chromatin and the spindle during cytokinesis. Taken together, our data reveal novel, important roles of GAS2L3 for faithful cell division. Our work thus contributes to the understanding of how DREAM regulates cytokinesis.
[Show abstract][Hide abstract] ABSTRACT: Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.
[Show abstract][Hide abstract] ABSTRACT: Since its first description by Theodor Boveri in 1888, the centrosome has been studied intensely, and it revealed detailed information about its structure, molecular composition and its various functions. The centrosome consists of two centrioles, which generally appear in electron microscopy as barrel-shaped structures usually composed of nine microtubule triplets. An amorphous mass of pericentriolar material surrounds the centrioles and accumulates many proteins important for the integrity and function of centrosomes, such as the γ-tubulin ring complex (γ-TuRC) that mediates microtubule nucleation and capping. In animal somatic cells, the centrosome generally accounts for the major microtubule organizing center, and the duplicated pair of centrosomes determines the poles of the microtubule-based mitotic spindle. Despite detailed insights into the centrosome's structure and function, it has been a complete mystery until a few years ago how centrosomes duplicate and assemble. Moreover, it is still largely unclear if and how centrosomal proteins or protein complexes are exchanged, replaced or qualitatively altered. Previously identified cytoplasmic granules, named "pericentriolar" or "centriolar satellites", might fulfil such functions in protein targeting and exchange, and communication between the centrosomes and the cytoplasm. In this review, we summarize current knowledge about the structure, molecular composition and possible roles of the satellites that seem to surround the core of the centrosome in most animal cells.
European journal of cell biology 09/2011; 90(12):983-9. DOI:10.1016/j.ejcb.2011.07.007 · 3.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Spatial separation of eukaryotic cells into the nuclear and cytoplasmic compartment permits uncoupling of DNA transcription from translation of mRNAs and allows cells to modify newly transcribed pre mRNAs extensively. Intronic sequences (introns), which interrupt the coding elements (exons), are excised ("spliced") from pre-mRNAs in the nucleus to yield mature mRNAs. This not only enables alternative splicing as an important source of proteome diversity, but splicing is also an essential process in all eukaryotes and knock-out or knock-down of splicing factors frequently results in defective cell proliferation and cell division. However, higher eukaryotes progress through cell division only after breakdown of the nucleus ("open mitosis"). Open mitosis suppresses basic nuclear functions such as transcription and splicing, but allows separate, mitotic functions of nuclear proteins in cell division. Mitotic defects arising after loss-of-function of splicing proteins therefore could be an indirect consequence of compromised splicing in the closed nucleus of the preceding interphase or reflect a direct contribution of splicing proteins to open mitosis. Although experiments to directly distinguish between these two alternatives have not been reported, indirect evidence exists for either hypotheses. In this review, we survey published data supporting an indirect function of splicing in open mitosis or arguing for a direct function of spliceosomal proteins in cell division.
[Show abstract][Hide abstract] ABSTRACT: The combination of RNA interference (RNAi) with the tetracycline-controlled transcription activation (tet) system promises to become a powerful method for conditional gene inactivation in cultured cells and in whole organisms. Here, we tested critical sequence elements that originated from miRNA mR-30 for optimal efficiency of RNAi-based gene knockdown in mammalian cells. Rationally designed miRNAs, expressed conditionally via the tet system, led to an efficient knockdown of the expression of both reporter genes and the endogenous mitotic spindle protein TPX2 in HeLa cells. Quantitative studies of the tet-controlled gene inactivation revealed that the residual expression of the target gene is an intrinsic attribute of all cells that cannot be eliminated either by increasing the miRNA to target mRNA ratio or by simultaneous expression of miRNAs targeting different sequences within the transcript. The kinetic analysis of the reversibility of the miRNA mediated knockdown suggests that the recovery of target gene expression is primarily driven by cell division. Our miRNA design provides a useful tool for conditional gene inactivation in combination with the RNA-polymerase II based tet system. The identified characteristics of the conditional RNAi-mediated knockdown need to be considered for its application in cell culture or in vivo.
Nucleic Acids Research 09/2010; 38(17):e168. DOI:10.1093/nar/gkq616 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The importin-beta-like transport receptors and RanGTP govern selective transport of proteins into the nucleus. It has now been shown that importin-beta2 (alternatively called transportin1) also selectively targets the motor protein Kif17 to primary cilia. In analogy to the nucleus, RanGTP in the intraciliary compartment mediates dissociation of Kif17 from its transport receptor and thereby completes import.