scholarly journals The Centrosomal Swiss Army Knife: A combined in silico and in vivo approach to the structure-function annotation of SPD-2 provides mechanistic insight into its functional diversity

2021 ◽  
Author(s):  
Mikaela Murph ◽  
Shaneen Singh ◽  
Mara Schvarzstein
Keyword(s):  
Structure Function ◽  
Cell Biology ◽  
Asymmetric Cell Division ◽  
Tumor Development ◽  
Coiled Coil ◽  
Immunoglobulin Superfamily ◽  
Major Sperm Protein ◽  
Developmental Abnormalities ◽  
Centriole Duplication

AbstractCentrosomes are organelles that function as hubs of microtubule nucleation and organization, with key roles in organelle positioning, asymmetric cell division, and ciliogenesis. Aberrant centrosome structure or function is linked to neurodegenerative diseases, developmental abnormalities, ciliopathies, and tumor development. A major regulator of centrosome biogenesis and function in C. elegans is the highly conserved protein Spindle-defective protein 2 (SPD-2), a homolog of the human CEP-192 protein. CeSPD-2 is required for centrosome maturation, centriole duplication, spindle assembly and cell polarity establishment. Despite its importance, the specific molecular mechanism of CeSPD-2 function is poorly understood. To address this gap in knowledge, we combined computational analysis with cell biology approaches to uncover structure-function relationships of CeSPD-2 that may shed mechanistic light on its function. Domain prediction analysis corroborated and refined previously identified coiled-coils and ASH (Aspm-SPD-2 Hydin) domains and identified new domains and motifs: an additional coiled-coil, a GEF domain, an Ig-like domain, and a PDZ-like domain. Our findings suggest that ASH domain belongs to the same superfold as PapD chaperone domains and Major Sperm Protein (MSP) domains within the larger Immunoglobulin superfamily. We have identified a large novel basic region in the CeSPD-2 ASH domain that harbors most of the predicted protein and nucleic acid contact residues in the ASH domain. In vivo, ASH::GFP localized to centrosomes and centrosome associated microtubules, and forms aggregates in the cytosol when overexpressed. This study lays the groundwork for designing rational hypothesis-based experiments for future analyses to further elaborate the mechanisms of CeSPD-2 function in vivo.

A vector system for fast-forward studies of the HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome in the model plant Nicotiana benthamiana

2021 ◽  
Author(s):  
Adeline Harant ◽  
Hsuan Pai ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
Vector System ◽  
In Planta ◽  
Functional Studies

Abstract Nicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis thaliana. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of nucleotide-binding leucine-rich repeat (NLR) plant immune receptors has greatly advanced following the discovery of the Arabidopsis HOPZ-ACTIVATED RESISTANCE1 (ZAR1) resistosome. Here, we describe a vector system of 72 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs (CC-NLRs) for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. Effector-activated and autoactive NbZAR1 trigger the cell death response in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana, thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source pZA plasmid collection forms an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals The Centrosomal, Putative Tumor Suppressor Protein TACC2 Is Dispensable for Normal Development, and Deficiency Does Not Lead to Cancer

2004 ◽  
Vol 24 (14) ◽  
pp. 6403-6409 ◽  
Author(s):  
Michael M. Schuendeln ◽  
Roland P. Piekorz ◽  
Christian Wichmann ◽  
Youngsoo Lee ◽  
Peter J. McKinnon ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Cell Cycle Progression ◽  
Tumor Development ◽  
Coiled Coil ◽  
Physiological Role ◽  
Tumor Suppressor Protein ◽  
Wild Type ◽  
Mouse Development

ABSTRACT TACC2 is a member of the transforming acidic coiled-coil-containing protein family and is associated with the centrosome-spindle apparatus during cell cycling. In vivo, the TACC2 gene is expressed in various splice forms predominantly in postmitotic tissues, including heart, muscle, kidney, and brain. Studies of human breast cancer samples and cell lines suggest a putative role of TACC2 as a tumor suppressor protein. To analyze the physiological role of TACC2, we generated mice lacking TACC2. TACC2-deficient mice are viable, develop normally, are fertile, and lack phenotypic changes compared to wild-type mice. Furthermore, TACC2 deficiency does not lead to an increased incidence of tumor development. Finally, in TACC2-deficient embryonic fibroblasts, proliferation and cell cycle progression as well as centrosome numbers are comparable to those in wild-type cells. Therefore, TACC2 is not required, nonredundantly, for mouse development and normal cell proliferation and is not a tumor suppressor protein.

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 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 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.

scholarly journals CD155 Promotes the Progression of Cervical Cancer Cells Through AKT/mTOR and NF-κB Pathways

2021 ◽  
Vol 11 ◽  
Author(s):  
Lu Liu ◽  
Ying Wang ◽  
Chen Geng ◽  
Aihong Wang ◽  
Sai Han ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Tumor Development ◽  
Tumor Formation ◽  
Immunoglobulin Superfamily ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Array ◽  
Cervical Lesions ◽  
Cervical Cancer Cells

Expression of the immunoglobulin superfamily member CD155 was increased in a variety of human malignancies, but the role of CD155 in tumorigenesis and tumor development in cervical cancer has not been elucidated. In this study, immunohistochemistry and enzyme-linked immunosorbent assay analyses showed that CD155 expression gradually increases with the degree of cervical lesions. In vitro and in vivo, reducing the expression of CD155 inhibited cell proliferation, cell viability and tumor formation and arrested the cell cycle in G0/G1 phase. Antibody array-based profiling of protein phosphorylation revealed that CD155 knockdown can inhibited the AKT/mTOR/NF-κB pathway and activated autophagy and apoptosis; the opposite effects were observed upon CD155 has overexpression. We proved that there is an interaction between CD155 and AKT by immunoprecipitation. We further confirmed the mechanism between CD155 and AKT/mTOR/NF-κB through rescue experiments. AKT knockdown reversed the anti-apoptotic effects and activation of the AKT/mTOR/NF-κB pathway induced by CD155 overexpression. Our research demonstrated that CD155 can interact with AKT to form a complex, activates the AKT/mTOR/NF-κB pathway and inhibit autophagy and apoptosis. Thus, CD155 is a potential screening and therapeutic biomarker for cervical cancer.

scholarly journals A vector system for fast-forward in vivo studies of the ZAR1 resistosome in the model plant Nicotiana benthamiana

2020 ◽  
Author(s):  
Adeline Harant ◽  
Toshiyuki Sakai ◽  
Sophien Kamoun ◽  
Hiroaki Adachi
Keyword(s):  
Nicotiana Benthamiana ◽  
Cell Biology ◽  
Plant Immunity ◽  
Transient Gene Expression ◽  
Coiled Coil ◽  
Experimental System ◽  
In Vivo Studies ◽  
Vector System ◽  
In Planta

ABSTRACTNicotiana benthamiana has emerged as a complementary experimental system to Arabidopsis. It enables fast-forward in vivo analyses primarily through transient gene expression and is particularly popular in the study of plant immunity. Recently, our understanding of NLR plant immune receptors has greatly advanced following the discovery of Arabidopsis ZAR1 resistosome. Here, we describe a novel vector system of 52 plasmids that enables functional studies of the ZAR1 resistosome in N. benthamiana. We showed that ZAR1 stands out among the coiled coil class of NLRs for being highly conserved across distantly related dicot plant species and confirmed NbZAR1 as the N. benthamiana ortholog of Arabidopsis ZAR1. NbZAR1 triggers autoimmune cell death in N. benthamiana and this activity is dependent on a functional N-terminal α1 helix. C-terminally tagged NbZAR1 remains functional in N. benthamiana thus enabling cell biology and biochemical studies in this plant system. We conclude that the NbZAR1 open source plasmids form an additional experimental system to Arabidopsis for in planta resistosome studies.

scholarly journals Direct membrane binding and self-interaction contribute to Mmr1 function in mitochondrial inheritance

2018 ◽  
Vol 29 (19) ◽  
pp. 2346-2357 ◽  
Author(s):  
WeiTing Chen ◽  
Holly A. Ping ◽  
Laura L. Lackner
Keyword(s):  
Structure Function ◽  
Membrane Binding ◽  
Coiled Coil ◽  
Mitochondrial Inheritance ◽  
Binding Assays ◽  
Membrane Interaction ◽  
Coiled Coil Domain ◽  
Necessary And Sufficient

Mitochondrial transport and anchoring mechanisms work in concert to position mitochondria to meet cellular needs. In yeast, Mmr1 functions as a mitochondrial adaptor for Myo2 to facilitate actin-based transport of mitochondria to the bud. Posttransport, Mmr1 is proposed to anchor mitochondria at the bud tip. Although both functions require an interaction between Mmr1 and mitochondria, the molecular basis of the Mmr1–mitochondria interaction is poorly understood. Our in vitro phospholipid binding assays indicate Mmr1 can directly interact with phospholipid membranes. Through structure–function studies we identified an unpredicted membrane-binding domain composed of amino acids 76–195 that is both necessary and sufficient for Mmr1 to interact with mitochondria in vivo and liposomes in vitro. In addition, our structure–function analyses indicate that the coiled-coil domain of Mmr1 is necessary and sufficient for Mmr1 self-interaction and facilitates the polarized localization of the protein. Disrupting either the Mmr1–membrane interaction or Mmr1 self-interaction leads to defects in mitochondrial inheritance. Therefore, direct membrane binding and self-interaction are necessary for Mmr1 function in mitochondrial inheritance and are utilized as a means to spatially and temporally regulate mitochondrial positioning.

Scanning Tunneling Microscopy and Atomic Force Microscopy of Enzymes and Enzyme Complexes

1990 ◽  
Vol 48 (3) ◽  
pp. 174-175
Author(s):  
Ronald D. Edstrom ◽  
Xiuru Yang ◽  
Mary E. Gurnack ◽  
Marcia A. Miller ◽  
Rui Yang ◽  
...  
Keyword(s):  
Cell Biology ◽  
Inorganic Ions ◽  
Bulk Liquid ◽  
Soluble Form ◽  
Scanning Tunneling ◽  
Tunneling Microscopy ◽  
Metabolic Channeling ◽  
Simplistic Notion ◽  
Soluble Enzymes

Many of the questions in biochemistry and cell biology are concerned with the relationships of proteins and other macromolecules in complex arrays which are responsible for carrying out metabolic sequences. The simplistic notion that the enzymes we isolate in soluble form from the cytoplasm were also soluble in vivo is being replaced by the concept that these enzymes occur in organized systems within the cell. In this newer view, the cytoplasm is organized and the “soluble enzymes” are in fact fixed in the cellular space and the only soluble components of the cell are small metabolites, inorganic ions etc. Further support for the concept of metabolic organization is provided by the evidence of metabolic channeling. It has been shown that for some metabolic pathways, the intermediates are not in free diffusion equilibrium with the bulk liquid in the cell but are passed along, more or less directly, from one enzyme to the next.

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