scholarly journals CP110 exhibits novel regulatory activities during centriole assembly in Drosophila

2013 ◽  
Vol 203 (5) ◽  
pp. 785-799 ◽  
Author(s):  
Anna Franz ◽  
Hélio Roque ◽  
Saroj Saurya ◽  
Jeroen Dobbelaere ◽  
Jordan W. Raff
Keyword(s):  
Cell Division ◽  
Centriole Duplication ◽  
Cilia Formation ◽  
Centriole Assembly

CP110 is a conserved centriole protein implicated in the regulation of cell division, centriole duplication, and centriole length and in the suppression of ciliogenesis. Surprisingly, we report that mutant flies lacking CP110 (CP110Δ) were viable and fertile and had no obvious defects in cell division, centriole duplication, or cilia formation. We show that CP110 has at least three functions in flies. First, it subtly influences centriole length by counteracting the centriole-elongating activity of several centriole duplication proteins. Specifically, we report that centrioles are ∼10% longer than normal in CP110Δ mutants and ∼20% shorter when CP110 is overexpressed. Second, CP110 ensures that the centriolar microtubules do not extend beyond the distal end of the centriole, as some centriolar microtubules can be more than 50 times longer than the centriole in the absence of CP110. Finally, and unexpectedly, CP110 suppresses centriole overduplication induced by the overexpression of centriole duplication proteins. These studies identify novel and surprising functions for CP110 in vivo in flies.

scholarly journals The Caenorhabditis elegans protein SAS-5 forms large oligomeric assemblies critical for centriole formation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Kacper B Rogala ◽  
Nicola J Dynes ◽  
Georgios N Hatzopoulos ◽  
Jun Yan ◽  
Sheng Kai Pong ◽  
...  
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Structural Analysis ◽  
Coiled Coil ◽  
Higher Order ◽  
Molecular Architecture ◽  
Eukaryotic Evolution ◽  
Centriole Duplication ◽  
Functional Implications

Centrioles are microtubule-based organelles crucial for cell division, sensing and motility. In Caenorhabditis elegans, the onset of centriole formation requires notably the proteins SAS-5 and SAS-6, which have functional equivalents across eukaryotic evolution. Whereas the molecular architecture of SAS-6 and its role in initiating centriole formation are well understood, the mechanisms by which SAS-5 and its relatives function is unclear. Here, we combine biophysical and structural analysis to uncover the architecture of SAS-5 and examine its functional implications in vivo. Our work reveals that two distinct self-associating domains are necessary to form higher-order oligomers of SAS-5: a trimeric coiled coil and a novel globular dimeric Implico domain. Disruption of either domain leads to centriole duplication failure in worm embryos, indicating that large SAS-5 assemblies are necessary for function in vivo.

scholarly journals The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Matthew A Cottee ◽  
Nadine Muschalik ◽  
Steven Johnson ◽  
Joanna Leveson ◽  
Jordan W Raff ◽  
...  
Keyword(s):  
Point Mutations ◽  
Coiled Coil ◽  
Higher Order ◽  
Centriole Duplication ◽  
Terminal Domain ◽  
Coiled Coil Domain ◽  
Centriole Assembly ◽  
Tetramer Structure

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

scholarly journals Crystal structures of the CPAP/STIL complex reveal its role in centriole assembly and human microcephaly

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Matthew A Cottee ◽  
Nadine Muschalik ◽  
Yao Liang Wong ◽  
Christopher M Johnson ◽  
Steven Johnson ◽  
...  
Keyword(s):  
Complex Formation ◽  
Crystal Structures ◽  
Brain Size ◽  
Point Mutations ◽  
Centriole Duplication ◽  
Structural Insight ◽  
Centriole Assembly ◽  
Cilia And Flagella ◽  
Insight Into

Centrioles organise centrosomes and template cilia and flagella. Several centriole and centrosome proteins have been linked to microcephaly (MCPH), a neuro-developmental disease associated with small brain size. CPAP (MCPH6) and STIL (MCPH7) are required for centriole assembly, but it is unclear how mutations in them lead to microcephaly. We show that the TCP domain of CPAP constitutes a novel proline recognition domain that forms a 1:1 complex with a short, highly conserved target motif in STIL. Crystal structures of this complex reveal an unusual, all-β structure adopted by the TCP domain and explain how a microcephaly mutation in CPAP compromises complex formation. Through point mutations, we demonstrate that complex formation is essential for centriole duplication in vivo. Our studies provide the first structural insight into how the malfunction of centriole proteins results in human disease and also reveal that the CPAP–STIL interaction constitutes a conserved key step in centriole biogenesis.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

scholarly journals Analysis of Cell Cycle and Replication of Mouse Macrophages afterIn VivoandIn VitroCryptococcus neoformans Infection Using Laser Scanning Cytometry

2012 ◽  
Vol 80 (4) ◽  
pp. 1467-1478 ◽  
Author(s):  
Carolina Coelho ◽  
Lydia Tesfa ◽  
Jinghang Zhang ◽  
Johanna Rivera ◽  
Teresa Gonçalves ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cytotoxic Effect ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Content Type ◽  
Cycle Arrest ◽  
Laser Scanning Cytometry

ABSTRACTWe investigated the outcome of the interaction ofCryptococcus neoformanswith murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis ofC. neoformanspromoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis ofC. neoformanspromoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellularC. neoformansresidence that manifested itself in impaired cell cycle completion as a consequence of a block in the G2/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replicationin vivoand demonstrated that these cells are capable of low levels of cell division in the presence or absence ofC. neoformansinfection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect ofC. neoformansinfection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferationin vivo.

scholarly journals Odorant receptors regulate the final glomerular coalescence of olfactory sensory neuron axons

2015 ◽  
Vol 112 (18) ◽  
pp. 5821-5826 ◽  
Author(s):  
Diego J. Rodriguez-Gil ◽  
Dianna L. Bartel ◽  
Austin W. Jaspers ◽  
Arie S. Mobley ◽  
Fumiaki Imamura ◽  
...  
Keyword(s):  
Cell Division ◽  
Sensory Neuron ◽  
Basal Cell ◽  
Olfactory Nerve ◽  
Olfactory Sensory Neuron ◽  
Odorant Receptors ◽  
Axon Extension ◽  
Mapping Strategy ◽  
Two Phases

Odorant receptors (OR) are strongly implicated in coalescence of olfactory sensory neuron (OSN) axons and the formation of olfactory bulb (OB) glomeruli. However, when ORs are first expressed relative to basal cell division and OSN axon extension is unknown. We developed an in vivo fate-mapping strategy that enabled us to follow OSN maturation and axon extension beginning at basal cell division. In parallel, we mapped the molecular development of OSNs beginning at basal cell division, including the onset of OR expression. Our data show that ORs are first expressed around 4 d following basal cell division, 24 h after OSN axons have reached the OB. Over the next 6+ days the OSN axons navigate the OB nerve layer and ultimately coalesce in glomeruli. These data provide a previously unidentified perspective on the role of ORs in homophilic OSN axon adhesion and lead us to propose a new model dividing axon extension into two phases. Phase I is OR-independent and accounts for up to 50% of the time during which axons approach the OB and begin navigating the olfactory nerve layer. Phase II is OR-dependent and concludes as OSN axons coalesce in glomeruli.

scholarly journals Asymmetric cell division-dominant neutral drift model for normal intestinal stem cell homeostasis

2019 ◽  
Vol 316 (1) ◽  
pp. G64-G74 ◽  
Author(s):  
Yoshitatsu Sei ◽  
Jianying Feng ◽  
Carson C. Chow ◽  
Stephen A. Wank
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Asymmetric Cell Division ◽  
Dominant Mode ◽  
Lineage Tracing ◽  
Intestinal Stem Cells ◽  
In Vivo Studies ◽  
Neutral Drift ◽  
Drift Model

The normal intestinal epithelium is continuously regenerated at a rapid rate from actively cycling Lgr5-expressing intestinal stem cells (ISCs) that reside at the crypt base. Recent mathematical modeling based on several lineage-tracing studies in mice shows that the symmetric cell division-dominant neutral drift model fits well with the observed in vivo growth of ISC clones and suggests that symmetric divisions are central to ISC homeostasis. However, other studies suggest a critical role for asymmetric cell division in the maintenance of ISC homeostasis in vivo. Here, we show that the stochastic branching and Moran process models with both a symmetric and asymmetric division mode not only simulate the stochastic growth of the ISC clone in silico but also closely fit the in vivo stem cell dynamics observed in lineage-tracing studies. In addition, the proposed model with highest probability for asymmetric division is more consistent with in vivo observations reported here and by others. Our in vivo studies of mitotic spindle orientations and lineage-traced progeny pairs indicate that asymmetric cell division is a dominant mode used by ISCs under normal homeostasis. Therefore, we propose the asymmetric cell division-dominant neutral drift model for normal ISC homeostasis. NEW & NOTEWORTHY The prevailing mathematical model suggests that intestinal stem cells (ISCs) divide symmetrically. The present study provides evidence that asymmetric cell division is the major contributor to ISC maintenance and thus proposes an asymmetric cell division-dominant neutral drift model. Consistent with this model, in vivo studies of mitotic spindle orientation and lineage-traced progeny pairs indicate that asymmetric cell division is the dominant mode used by ISCs under normal homeostasis.

scholarly journals The 3D architecture and molecular foundations of de novo centriole assembly via bicentrioles

2020 ◽  
Author(s):  
Sónia Gomes Pereira ◽  
Ana Laura Sousa ◽  
Catarina Nabais ◽  
Tiago Paixão ◽  
Alexander. J. Holmes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Division ◽  
De Novo ◽  
Cellular Functions ◽  
Spindle Poles ◽  
3D Architecture ◽  
Centriole Assembly ◽  
Work Supports ◽  
Wall Components

Abstract/SummaryCentrioles are structurally conserved organelles, composing both centrosomes and cilia. In animal cycling cells, centrioles often form through a highly characterized process termed canonical duplication. However, a large diversity of eukaryotes form centrioles de novo through uncharacterized pathways. This unexplored diversity is key to understanding centriole assembly mechanisms and how they evolved to assist specific cellular functions. Here, combining electron microscopy and tomography, we show that during spermatogenesis of the moss Physcomitrium patens, centrioles are born as a co-axially oriented centriole pair united by a cartwheel. We observe that microtubules emanate from those bicentrioles, which localize to the spindle poles during cell division. Thereafter, each bicentriole breaks apart, and the two resulting sister centrioles mature asymmetrically, elongating specific microtubule triplets and a naked cartwheel. Subsequently, two cilia are assembled which are capable of beating asynchronously. We further show that conserved cartwheel and centriole wall components, SAS6, BLD10 and POC1 are expressed during spermatogenesis and are required for this de novo biogenesis pathway. Our work supports a scenario where centriole biogenesis is more diverse than previously thought and that conserved molecular modules underlie diversification of this essential pathway.

scholarly journals Synthesis of Novel Pyran Fragments to Incorporate into Peloruside Analogues

2021 ◽  
Author(s):  
◽  
Oliver Bayley
Keyword(s):  
Cell Division ◽  
Scale Up ◽  
Great Promise ◽  
Peloruside A ◽  
Bioactive Secondary Metabolite ◽  
Mycale Hentscheli ◽  
Significant Attention ◽  
Simple Carbohydrate

<p>Cancer is currently the second largest cause of death globally, leading to a high demand for new and effective chemotherapeutics. For years, natural products have been used as a source of new bioactive compounds; of particular interest in this context, as a source of new chemotherapeutics. One chemotherapeutic candidate which has attracted significant attention in synthetic and medicinal chemistry communities, is peloruside A. Peloruside A is a bioactive secondary metabolite isolated from the New Zealand marine sponge Mycale hentscheli. Since its discovery, peloruside A has shown great promise in cancer studies both in vivo and in vitro with effects observed even at nanomolar concentrations. These chemotherapeutic effects have been shown to occur by halting cell division at the G2/M checkpoint via microtubule stabilisation. Of particular interest is that this stabilisation occurs in a manner distinct from that of the already established taxane class of microtubule stabilising drugs. This means that peloruside A is able to offer both inhibition of cell division in Taxol® resistant cells and synergistic inhibition alongside the current taxane drugs. Since peloruside A is not abundantly available from its natural source, there is a strong incentive for the development of new synthetic strategies for peloruside A production. Unfortunately attempts at aquaculture and attempts at developing an industrial scale synthesis have both proven unsuccessful thus far. In an attempt to overcome some of the difficulties with the scale up of peloruside, analogues have been developed that are intended to have similar bioactivity to peloruside A but simpler, more concise, synthetic routes. These analogues will also enable further elucidation of the binding properties of peloruside A. This project focuses on the generation of a functionalised pyran fragment, starting from a simple carbohydrate, that may be incorporated into the proposed analogues.</p>

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