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 Hydrophobicity variations along the surface of the coiled-coil rod may mediate striated muscle myosin assembly in Caenorhabditis elegans.

1996 ◽  
Vol 135 (2) ◽  
pp. 371-382 ◽  
Author(s):  
P E Hoppe ◽  
R H Waterston
Keyword(s):  
Caenorhabditis Elegans ◽  
Striated Muscle ◽  
Coiled Coil ◽  
Thick Filament ◽  
Surface Hydrophobicity ◽  
Filament Assembly ◽  
Thick Filaments ◽  
Characteristic Variation ◽  
Muscle Myosin

Caenorhabditis elegans body wall muscle contains two isoforms of myosin heavy chain, MHC A and MHC B, that differ in their ability to initiate thick filament assembly. Whereas mutant animals that lack the major isoform, MHC B, have fewer thick filaments, mutant animals that lack the minor isoform, MHC A, contain no normal thick filaments. MHC A, but not MHC B, is present at the center of the bipolar thick filament where initiation of assembly is thought to occur (Miller, D.M.,I. Ortiz, G.C. Berliner, and H.F. Epstein. 1983. Cell. 34:477-490). We mapped the sequences that confer A-specific function by constructing chimeric myosins and testing them in vivo. We have identified two distinct regions of the MHC A rod that are sufficient in chimeric myosins for filament initiation function. Within these regions, MHC A displays a more hydrophobic rod surface, making it more similar to paramyosin, which forms the thick filament core. We propose that these regions play an important role in filament initiation, perhaps mediating close contacts between MHC A and paramyosin in an antiparallel arrangement at the filament center. Furthermore, our analysis revealed that all striated muscle myosins show a characteristic variation in surface hydrophobicity along the length of the rod that may play an important role in driving assembly and determining the stagger at which dimers associate.

scholarly journals Dissociating the Centrosomal Matrix Protein AKAP450 from Centrioles Impairs Centriole Duplication and Cell Cycle Progression

2003 ◽  
Vol 14 (6) ◽  
pp. 2436-2446 ◽  
Author(s):  
Guy Keryer ◽  
Oliwia Witczak ◽  
Annie Delouvée ◽  
Wolfram A. Kemmner ◽  
Danielle Rouillard ◽  
...  
Keyword(s):  
Cell Division ◽  
Hela Cells ◽  
Cellular Localization ◽  
Cell Cycle Progression ◽  
Matrix Protein ◽  
Coiled Coil ◽  
Cell Division Cycle ◽  
Scaffolding Protein ◽  
Centriole Duplication ◽  
C Terminus

Centrosomes provide docking sites for regulatory molecules involved in the control of the cell division cycle. The centrosomal matrix contains several proteins, which anchor kinases and phosphatases. The large A-Kinase Anchoring Protein AKAP450 is acting as a scaffolding protein for other components of the cell signaling machinery. We selectively perturbed the centrosome by modifying the cellular localization of AKAP450. We report that the expression in HeLa cells of the C terminus of AKAP450, which contains the centrosome-targeting domain of AKAP450 but not its coiled-coil domains or binding sites for signaling molecules, leads to the displacement of the endogenous centrosomal AKAP450 without removing centriolar or pericentrosomal components such as centrin, γ-tubulin, or pericentrin. The centrosomal protein kinase A type II α was delocalized. We further show that this expression impairs cytokinesis and increases ploidy in HeLa cells, whereas it arrests diploid RPE1 fibroblasts in G1, thus further establishing a role of the centrosome in the regulation of the cell division cycle. Moreover, centriole duplication is interrupted. Our data show that the association between centrioles and the centrosomal matrix protein AKAP450 is critical for the integrity of the centrosome and for its reproduction.

scholarly journals The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling

2017 ◽  
Vol 28 (23) ◽  
pp. 3298-3314 ◽  
Author(s):  
Shruthi Viswanath ◽  
Massimiliano Bonomi ◽  
Seung Joong Kim ◽  
Vadim A. Klenchin ◽  
Keenan C. Taylor ◽  
...  
Keyword(s):  
Cell Biology ◽  
Spindle Pole Body ◽  
Coiled Coil ◽  
Resonance Energy ◽  
Spindle Pole ◽  
Physical Structure ◽  
Molecular Architecture ◽  
X Ray ◽  
Pole Body

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

scholarly journals Phytobacter diazotrophicus from Intestine of Caenorhabditis elegans Confers Colonization-Resistance against Bacillus nematocida Using Flagellin (FliC) as an Inhibition Factor

Pathogens ◽  
2022 ◽  
Vol 11 (1) ◽  
pp. 82
Author(s):  
Qiuhong Niu ◽  
Suyao Liu ◽  
Mingshen Yin ◽  
Shengwei Lei ◽  
Fabio Rezzonico ◽  
...  
Keyword(s):  
Cell Division ◽  
Caenorhabditis Elegans ◽  
Membrane Protein ◽  
Bacterial Cells ◽  
Colonization Resistance ◽  
Flic Gene ◽  
C Elegans ◽  
Free Living Nematode ◽  
Bacterium Bacillus

Symbiotic microorganisms in the intestinal tract can influence the general fitness of their hosts and contribute to protecting them against invading pathogens. In this study, we obtained isolate Phytobacter diazotrophicus SCO41 from the gut of free-living nematode Caenorhabditis elegans that displayed strong colonization-resistance against invading biocontrol bacterium Bacillus nematocida B16. The colonization-resistance phenotype was found to be mediated by a 37-kDa extracellular protein that was identified as flagellin (FliC). With the help of genome information, the fliC gene was cloned and heterologously expressed in E. coli. It could be shown that the B. nematocida B16 grows in chains rather than in planktonic form in the presence of FliC. Scanning Electronic Microscopy results showed that protein FliC-treated B16 bacterial cells are thinner and longer than normal cells. Localization experiments confirmed that the protein FliC is localized in both the cytoplasm and the cell membrane of B16 strain, in the latter especially at the position of cell division. ZDOCK analysis showed that FliC could bind with serine/threonine protein kinase, membrane protein insertase YidC and redox membrane protein CydB. It was inferred that FliC interferes with cell division of B. nematocidal B16, therefore inhibiting its colonization of C. elegans intestines in vivo. The isolation of P. diazotrophicus as part of the gut microbiome of C. elegans not only provides interesting insights about the lifestyle of this nitrogen-fixing bacterium, but also reveals how the composition of the natural gut microbiota of nematodes can affect biological control efforts by protecting the host from its natural enemies.

scholarly journals Plant Kinesin-12: Localization Heterogeneity and Functional Implications

2019 ◽  
Vol 20 (17) ◽  
pp. 4213 ◽  
Author(s):  
Sabine Müller ◽  
Pantelis Livanos
Keyword(s):  
Cell Division ◽  
Current Knowledge ◽  
Male Gametophyte ◽  
Domain Architecture ◽  
Coiled Coil ◽  
Cell Plate ◽  
Cellular Level ◽  
Functional Diversification ◽  
Functional Implications ◽  
Directed Motility

Kinesin-12 family members are characterized by an N-terminal motor domain and the extensive presence of coiled-coil domains. Animal orthologs display microtubule plus-end directed motility, bundling of parallel and antiparallel microtubules, plus-end stabilization, and they play a crucial role in spindle assembly. In plants, kinesin-12 members mediate a number of developmental processes including male gametophyte, embryo, seedling, and seed development. At the cellular level, they participate in critical events during cell division. Several kinesin-12 members localize to the phragmoplast midzone, interact with isoforms of the conserved microtubule cross-linker MICROTUBULE-ASSOCIATED PROTEIN 65 (MAP65) family, and are required for phragmoplast stability and expansion, as well as for proper cell plate development. Throughout cell division, a subset of kinesin-12 reside, in addition or exclusively, at the cortical division zone and mediate the accurate guidance of the phragmoplast. This review aims to summarize the current knowledge on kinesin-12 in plants and shed some light onto the heterogeneous localization and domain architecture, which potentially conceals functional diversification.

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.

Intermediate filaments: molecular architecture, assembly, dynamics and polymorphism

1999 ◽  
Vol 32 (2) ◽  
pp. 99-187 ◽  
Author(s):  
David A. D. Parry ◽  
Peter M. Steinert
Keyword(s):  
Intermediate Filaments ◽  
Intermediate Filament ◽  
Tertiary Structure ◽  
Quaternary Structure ◽  
Lattice Structure ◽  
Coiled Coil ◽  
Salt Bridges ◽  
Molecular Architecture

1. Introduction 1002. Molecular architecture 1072.1 Primary structure 1082.1.1 Homologous regions 1092.1.2 Chain typing 1152.1.3 Post-translational modifications 1172.2 Secondary structure 1182.2.1 Central rod domain 1182.2.2 Head and tail domains 1192.3 Tertiary structure 1232.3.1 Coiled-coil rod domain 1232.3.1.1 Specificity through salt bridges 1242.3.1.2 Specificity through apolar interactions 1272.3.1.3 A consensus trigger sequence for two-stranded coiled-coils 1282.3.2 Discontinuities in the rod domain 1282.3.2.1 Links 1292.3.2.2 Stutter 1312.3.3 Head and tail domains 1312.4 Electron microscope observations 1333. Assembly 1363.1 Role of the coiled-coil rod domain 1373.2 Role of end domains 1413.3 Experimentally induced crosslinks and modes of assembly 1453.4 Naturally occurring crosslinks for tissue coordination 1543.5 STEM data 1544. Quaternary structure 1604.1 Protofilaments and protofibrils 1604.2 Head and tail domains 1634.3 Surface lattice structure 1644.4 Crystal studies on intermediate filament fragments 1685. Polymorphism 1695.1 Variations on a theme 1705.1.1 Axial structure 1705.1.2 Lateral structure 1716. Keratin intermediate filament chains in diseases 1727. Concluding remarks 1758. Acknowledgments 1769. References 176Three types of intracellular filament have been identified in eukaryotic cells, and together they constitute the key elements of the cytoskeleton. They are the microtubules, the actin-containing microfilaments and the intermediate filaments. The uniqueness of the former two types of filament in cells has been well known for a long time but, in contrast, the intermediate filaments have been a relative new-comer to the scene. The microtubules and the microfilaments have always been easy to distinguish from one another on the grounds of their respective sizes (microtubules are about 25 nm in diameter and microfilaments are about 7–10 nm in diameter). Additionally, microtubules were capable of being disaggregated by the action of colchicine, and microfilaments could be disassembled by other drugs or be decorated with heavy meromyosin to generate arrowhead-like structures. Importantly, in both microtubules and microfilaments the constituent protein subunits were arranged to give the filaments a directionality, and the ability of these filaments to function in vivo depended crucially on this feature of their structure. Microtubules, for example, are involved in mitosis, motility and transport within the cell: each of these functions is clearly a ‘directional’ one. With this background the discovery and characterization of the intermediate filaments can begin.

scholarly journals The human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments

2018 ◽  
Author(s):  
Luke A. Helgeson ◽  
Alex Zelter ◽  
Michael Riffle ◽  
Michael J. MacCoss ◽  
Charles L. Asbury ◽  
...  
Keyword(s):  
Cell Division ◽  
Optical Tweezers ◽  
Coiled Coil ◽  
Microtubule Binding ◽  
C Terminus ◽  
Switching Dynamics ◽  
Microtubule Binding Domain ◽  
Ndc80 Complex ◽  
Bearing Assembly

ABSTRACTAccurate segregation of chromosomes relies on the force-bearing capabilities of the kinetochore to robustly attach chromosomes to dynamic microtubule tips. The human Ska complex and Ndc80 complex are outer-kinetochore components that bind microtubules and are required to fully stabilize kinetochore-microtubule attachments in vivo. While purified Ska complex tracks with disassembling microtubule tips, it remains unclear whether the Ska complex-microtubule interaction is sufficiently strong to make a significant contribution to kinetochore-microtubule coupling. Alternatively, Ska complex might affect kinetochore coupling indirectly, through recruitment of phospho-regulatory factors. Using optical tweezers, we show that the Ska complex itself bears load on microtubule tips, strengthens Ndc80 complex-based tip attachments, and increases the switching dynamics of the attached microtubule tips. Crosslinking mass spectrometry suggests the Ska complex directly binds Ndc80 complex through interactions between the Ska3 unstructured C-terminal region and the coiled-coil regions of each Ndc80 complex subunit. Deletion of the Ska complex microtubule-binding domain or the Ska3 C-terminus prevents Ska complex from strengthening Ndc80 complex-based attachments. Together our results indicate that the Ska complex can directly strengthen the kinetochore microtubule interface and regulate microtubule tip dynamics by forming an additional connection between the Ndc80 complex and the microtubule.SIGNIFICANCE STATEMENTMicrotubules are dynamic, tube-like structures that drive the segregation of duplicated chromosomes during cell division. The Ska complex is part of a molecular machine that forms force-bearing connections between chromosomes and microtubule ends. Depletion of the Ska complex destabilizes these connections and disrupts cell division. The Ska complex binds microtubules but it is unknown if it directly holds force at microtubules or indirectly stabilizes the connections. Here, we show that the Ska complex makes a direct force-bearing linkage with microtubule ends and assembles with another microtubule binding component, the Ndc80 complex, to strengthen its ability to withstand force. Our results suggest that the Ska and Ndc80 complexes work together to maintain the connections between chromosomes and microtubule ends.

scholarly journals STIL binding to Polo-box 3 of PLK4 regulates centriole duplication

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Christian Arquint ◽  
Anna-Maria Gabryjonczyk ◽  
Stefan Imseng ◽  
Raphael Böhm ◽  
Evelyn Sauer ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Coiled Coil ◽  
Binding Modes ◽  
Cycle Progression ◽  
Secondary Interaction ◽  
Centriole Duplication ◽  
Coiled Coil Region ◽  
In Vivo Analysis

Polo-like kinases (PLK) are eukaryotic regulators of cell cycle progression, mitosis and cytokinesis; PLK4 is a master regulator of centriole duplication. Here, we demonstrate that the SCL/TAL1 interrupting locus (STIL) protein interacts via its coiled-coil region (STIL-CC) with PLK4 in vivo. STIL-CC is the first identified interaction partner of Polo-box 3 (PB3) of PLK4 and also uses a secondary interaction site in the PLK4 L1 region. Structure determination of free PLK4-PB3 and its STIL-CC complex via NMR and crystallography reveals a novel mode of Polo-box–peptide interaction mimicking coiled-coil formation. In vivo analysis of structure-guided STIL mutants reveals distinct binding modes to PLK4-PB3 and L1, as well as interplay of STIL oligomerization with PLK4 binding. We suggest that the STIL-CC/PLK4 interaction mediates PLK4 activation as well as stabilization of centriolar PLK4 and plays a key role in centriole duplication.

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