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Journal of Clinical Immunology Feb 2015
Review
Topics: Animals; Autoimmune Diseases; Autoimmunity; Carrier Proteins; Disease Models, Animal; Genetic Association Studies; Humans; Immunologic Deficiency Syndromes; Leukemia; Mice; Mice, Knockout; Microfilament Proteins; Molecular Targeted Therapy; Mutation; Phenotype; Protein Binding; Protein Interaction Domains and Motifs
PubMed: 25666293
DOI: 10.1007/s10875-015-0130-z -
Proceedings of the National Academy of... Sep 2022To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and...
To orchestrate cell mechanics, trafficking, and motility, cytoskeletal filaments must assemble into higher-order networks whose local subcellular architecture and composition specify their functions. Cross-linking proteins bridge filaments at the nanoscale to control a network's μm-scale geometry, thereby conferring its mechanical properties and functional dynamics. While these interfilament linkages are key determinants of cytoskeletal function, their structural mechanisms remain poorly understood. Plastins/fimbrins are an evolutionarily ancient family of tandem calponin-homology domain (CHD) proteins required to construct multiple classes of actin networks, which feature diverse geometries specialized to power cytokinesis, microvilli and stereocilia biogenesis, and persistent cell migration. Here, we focus on the structural basis of actin network assembly by human T-plastin, a ubiquitously expressed isoform necessary for the maintenance of stable cellular protrusions generated by actin polymerization forces. By implementing a machine-learning-enabled cryo-electron microscopy pipeline for visualizing cross-linkers bridging multiple filaments, we uncover a sequential bundling mechanism enabling T-plastin to bridge pairs of actin filaments in both parallel and antiparallel orientations. T-plastin populates distinct structural landscapes in these two bridging orientations that are selectively compatible with actin networks featuring divergent architectures and functions. Our structural, biochemical, and cell biological data highlight inter-CHD linkers as key structural elements underlying flexible but stable cross-linking that are likely to be disrupted by T-plastin mutations that cause hereditary bone diseases.
Topics: Actin Cytoskeleton; Actins; Cryoelectron Microscopy; Humans; Membrane Glycoproteins; Microfilament Proteins; Polymerization
PubMed: 36067297
DOI: 10.1073/pnas.2205370119 -
Oncotarget Sep 2015
Topics: Actins; Animals; Cytoskeleton; Genes, Recessive; Humans; Mice; Mice, Knockout; Microfilament Proteins; Muscle Hypotonia; Muscle Proteins; Muscle, Skeletal; Mutation; Myopathies, Nemaline; Phenotype; Protein Binding; Xenopus
PubMed: 26337340
DOI: 10.18632/oncotarget.5267 -
Proceedings of the National Academy of... Jul 2022The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference...
The abLIM1 is a nonerythroid actin-binding protein critical for stable plasma membrane-cortex interactions under mechanical tension. Its depletion by RNA interference results in sparse, poorly interconnected cortical actin networks and severe blebbing of migrating cells. Its isoforms, abLIM-L, abLIM-M, and abLIM-S, contain, respectively four, three, and no LIM domains, followed by a C terminus entirely homologous to erythroid cortex protein dematin. How abLIM1 functions, however, remains unclear. Here we show that abLIM1 is a liquid-liquid phase separation (LLPS)-dependent self-organizer of actin networks. Phase-separated condensates of abLIM-S-mimicking ΔLIM or the major isoform abLIM-M nucleated, flew along, and cross-linked together actin filaments (F-actin) to produce unique aster-like radial arrays and interconnected webs of F-actin bundles. Interestingly, ΔLIM condensates facilitated actin nucleation and network formation even in the absence of Mg. Our results suggest that abLIM1 functions as an LLPS-dependent actin nucleator and cross-linker and provide insights into how LLPS-induced condensates could self-construct intracellular architectures of high connectivity and plasticity.
Topics: Actin Cytoskeleton; Actins; Humans; LIM Domain Proteins; Microfilament Proteins; Protein Isoforms; RNA Interference
PubMed: 35858327
DOI: 10.1073/pnas.2122420119 -
British Journal of Cancer Sep 2020The molecular signature underlying pancreatic ductal adenocarcinoma (PDAC) progression may include key proteins affecting the malignant phenotypes. Here, we aimed to...
BACKGROUND
The molecular signature underlying pancreatic ductal adenocarcinoma (PDAC) progression may include key proteins affecting the malignant phenotypes. Here, we aimed to identify the proteins implicated in PDAC with different tumour-node-metastasis (TNM) stages.
METHODS
Eight-plex isobaric tags coupled with two-dimensional liquid chromatography-tandem mass spectrometry were used to analyse the proteome of PDAC tissues with different TNM stages. A loss-of-function study was performed to evaluate the oncogenic roles of WD repeat-containing protein 1 (WDR1) in PDAC. The molecular mechanism by which WDR1 promotes PDAC progression was studied by real-time qPCR, Western blotting, proximity ligation assay and co-immunoprecipitation.
RESULTS
A total of 5036 proteins were identified, and 4708 proteins were quantified with high confidence. Compared with normal pancreatic tissues, 37 proteins were changed significantly in PDAC tissues of different stages. Moreover, 64 proteins were upregulated or downregulated in a stepwise manner as the TNM stages of PDAC increased, and 10 proteins were related to tumorigenesis. The functionally uncharacterised protein, WDR1, was highly expressed in PDAC and predicted a poor prognosis. WDR1 knockdown suppressed PDAC tumour growth and metastasis in vitro and in vivo. Moreover, WDR1 knockdown repressed the activity of the Wnt/β-Catenin pathway; ectopic expression of a stabilised form of β-Catenin restored the suppressive effects of WDR1 knockdown. Mechanistically, WDR1 interacted with USP7 to prevent ubiquitination-mediated degradation of β-Catenin.
CONCLUSION
Our study identifies several previous functional unknown proteins implicated in the progression of PDAC, and provides new insight into the oncogenic roles of WDR1 in PDAC development.
Topics: Animals; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Humans; Male; Mice; Microfilament Proteins; Pancreatic Neoplasms; Ubiquitin-Specific Peptidase 7; Ubiquitination; Wnt Signaling Pathway; beta Catenin
PubMed: 32601462
DOI: 10.1038/s41416-020-0929-0 -
Molecular Biology of the Cell Mar 2023Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60...
Clathrin-mediated endocytosis is a conserved eukaryotic membrane trafficking pathway that is driven by a sequentially assembled molecular machinery that contains over 60 different proteins. SH3 domains are the most abundant protein-protein interaction domain in this process, but the function of most SH3 domains in protein dynamics remains elusive. Using mutagenesis and live-cell fluorescence microscopy in the budding yeast , we dissected SH3-mediated regulation of the endocytic pathway. Our data suggest that multiple SH3 domains regulate the actin nucleation-promoting Las17-Vrp1 complex, and that the network of SH3 interactions coordinates both Las17-Vrp1 assembly and dissociation. Furthermore, most endocytic SH3 domain proteins use the SH3 domain for their own recruitment, while a minority use the SH3 domain to recruit other proteins and not themselves. Our results provide a dynamic map of SH3 functions in yeast endocytosis and a framework for SH3 interaction network studies across biology.
Topics: Saccharomyces cerevisiae; src Homology Domains; Saccharomyces cerevisiae Proteins; Microfilament Proteins; Actins; Endocytosis; Wiskott-Aldrich Syndrome Protein
PubMed: 36696224
DOI: 10.1091/mbc.E22-09-0406 -
International Journal of Molecular... May 2023The mammalian formin family comprises fifteen multi-domain proteins that regulate actin dynamics and microtubules in vitro and in cells. Evolutionarily conserved formin... (Review)
Review
The mammalian formin family comprises fifteen multi-domain proteins that regulate actin dynamics and microtubules in vitro and in cells. Evolutionarily conserved formin homology (FH) 1 and 2 domains allow formins to locally modulate the cell cytoskeleton. Formins are involved in several developmental and homeostatic processes, as well as human diseases. However, functional redundancy has long hampered studies of individual formins with genetic loss-of-function approaches and prevents the rapid inhibition of formin activities in cells. The discovery of small molecule inhibitor of formin homology 2 domains (SMIFH2) in 2009 was a disruptive change that provided a powerful chemical tool to explore formins' functions across biological scales. Here, I critically discuss the characterization of SMIFH2 as a pan-formin inhibitor, as well as growing evidence of unexpected off-target effects. By collating the literature and information hidden in public repositories, outstanding controversies and fundamental open questions about the substrates and mechanism of action of SMIFH2 emerge. Whenever possible, I propose explanations for these discrepancies and roadmaps to address the paramount open questions. Furthermore, I suggest that SMIFH2 be reclassified as a multi-target inhibitor for its appealing activities on proteins involved in pathological formin-dependent processes. Notwithstanding all drawbacks and limitations, SMIFH2 will continue to prove useful in studying formins in health and disease in the years to come.
Topics: Animals; Humans; Formins; Microfilament Proteins; Actins; Cytoskeleton; Biology; Actin Cytoskeleton; Mammals
PubMed: 37240404
DOI: 10.3390/ijms24109058 -
Proceedings of the National Academy of... Dec 2019Atomistic molecular dynamics simulations of concentrated protein solutions in the presence of a phospholipid bilayer are presented to gain insights into the dynamics and...
Atomistic molecular dynamics simulations of concentrated protein solutions in the presence of a phospholipid bilayer are presented to gain insights into the dynamics and interactions at the cytosol-membrane interface. The main finding is that proteins that are not known to specifically interact with membranes are preferentially excluded from the membrane, leaving a depletion zone near the membrane surface. As a consequence, effective protein concentrations increase, leading to increased protein contacts and clustering, whereas protein diffusion becomes faster near the membrane for proteins that do occasionally enter the depletion zone. Since protein-membrane contacts are infrequent and short-lived in this study, the structure of the lipid bilayer remains largely unaffected by the crowded protein solution, but when proteins do contact lipid head groups, small but statistically significant local membrane curvature is induced, on average.
Topics: Bacterial Proteins; Cell Membrane; Cluster Analysis; Diffusion; Lipid Bilayers; Microfilament Proteins; Molecular Dynamics Simulation; Phosphatidylcholines; Proteins; Sphingomyelins; Ubiquitin
PubMed: 31740611
DOI: 10.1073/pnas.1910771116 -
Pancreas 2019The pathogenesis of pancreatic neuroendocrine tumors (PNETs) is still unclear. We propose Frabin as a new molecular alteration in PNETs. Frabin is a guanine nucleotide...
OBJECTIVE
The pathogenesis of pancreatic neuroendocrine tumors (PNETs) is still unclear. We propose Frabin as a new molecular alteration in PNETs. Frabin is a guanine nucleotide exchange factor playing a role in mediating actin cytoskeleton changes during cell migration, morphogenesis, polarization, and division.
METHODS
Patients with PNETs of different grades were assessed for Frabin expression using immunohistochemistry and tissue microarray. The tissue microarray included 12 grade 1 and 3 grade 2 PNETs and 14 grade 3 pancreatic neuroendocrine carcinomas (PECAs). Frabin immunostain was scored with Allred system. Statistical analysis used SAS and R software. Immunohistochemistry scores were correlated with tumor grade and stage. The Spearman correlation coefficient was calculated with P values.
RESULTS
Pancreatic neuroendocrine tumors were graded according to the World Health Organization 2017 guidelines. Frabin was expressed by 24 (82.7%) of the PNET/PECA studied. Only 5 (17.2%) of the 29 PNETs/PECA evaluated were Frabin negative. Frabin expression was cytoplasmic in all cases. We found a significant positive correlation (ρ = 0.47) between Frabin immunohistochemistry score and tumor grade (P = 0.01). No correlation was found between Frabin expression and tumor stage (P = 0.91).
CONCLUSIONS
We report Frabin overexpression as a novel molecular alteration occurring in PNETs/PECAs.
Topics: Adult; Aged; Female; Humans; Immunohistochemistry; Male; Microfilament Proteins; Middle Aged; Neoplasm Grading; Neuroendocrine Tumors; Pancreatic Neoplasms
PubMed: 31688594
DOI: 10.1097/MPA.0000000000001422 -
European Journal of Cell Biology 2022The viscoelastic parameters of the cell can report on the cell state, cellular processes and diseases. Cell mechanics strongly rely on the properties of the...
The viscoelastic parameters of the cell can report on the cell state, cellular processes and diseases. Cell mechanics strongly rely on the properties of the cytoskeleton, an important system of subcellular filaments, especially on the high-level structures that actin forms together with actin-binding proteins (ABPs). In normal cells, components of the cytoskeleton are highly integrated, and their functions are well orchestrated. In contrast, impaired expression and functioning of ABPs lead to the increasing ability of cancer cells to resist chemotherapy and metastasize. ABP-mediated changes in the cytoskeleton architecture can lead to changes in the mechanical properties of the actin network, both locally and at the level of the whole cell. Until now, in cancer-related studies, mechanical data have been used less frequently, compared to biochemical tests or cell migration assays. Here, we will review current methods for analyzing the mechanical properties of cells and provide the available data on the contribution of ABPs in determining cell mechanical properties important for the investigation of cellular functions, particularly in cancers.
Topics: Actin Cytoskeleton; Actins; Cytoskeleton; Microfilament Proteins; Microtubules
PubMed: 35653881
DOI: 10.1016/j.ejcb.2022.151241