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Protein Science : a Publication of the... May 2023Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is...
Palladin is an actin binding protein that is specifically upregulated in metastatic cancer cells but also colocalizes with actin stress fibers in normal cells and is critical for embryonic development as well as wound healing. Of nine isoforms present in humans, only the 90 kDa isoform of palladin, comprising three immunoglobulin (Ig) domains and one proline-rich region, is ubiquitously expressed. Previous work has established that the Ig3 domain of palladin is the minimal binding site for F-actin. In this work, we compare functions of the 90 kDa isoform of palladin to the isolated actin binding domain. To understand the mechanism of action for how palladin can influence actin assembly, we monitored F-actin binding and bundling as well as actin polymerization, depolymerization, and copolymerization. Together, these results demonstrate that there are key differences between the Ig3 domain and full-length palladin in actin binding stoichiometry, polymerization, and interactions with G-actin. Understanding the role of palladin in regulating the actin cytoskeleton may help us develop means to prevent cancer cells from reaching the metastatic stage of cancer progression.
Topics: Humans; Actins; Cytoskeletal Proteins; Microfilament Proteins; Actin Cytoskeleton; Protein Isoforms; Phosphoproteins
PubMed: 37027210
DOI: 10.1002/pro.4638 -
STAR Protocols Dec 2022The unreliability of commercial recombinant asprosin preparations and variability between asprosin detection assays have proven to be a bottleneck in experimental...
The unreliability of commercial recombinant asprosin preparations and variability between asprosin detection assays have proven to be a bottleneck in experimental interpretation. This protocol describes the use of viral vectors and expression plasmid for overexpression and secretion of human asprosin to achieve sustained elevation of asprosin protein in mice and HEK293T cells without using recombinant proteins. This protocol also includes a sandwich ELISA using anti-asprosin monoclonal antibodies for detection of asprosin in media from cultured cells and in plasma of mice. For complete details on the use and execution of this protocol, please refer to Duerrschmid et al. (2017), Mishra et al. (2021), and Mishra et al. (2022).
Topics: Mice; Humans; Animals; HEK293 Cells; Peptide Fragments; Peptide Hormones; Microfilament Proteins; Fibrillin-1
PubMed: 36240062
DOI: 10.1016/j.xpro.2022.101762 -
Science Advances Jan 2023Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit...
Regulation of the Arp2/3 complex is required for productive nucleation of branched actin networks. An emerging aspect of regulation is the incorporation of subunit isoforms into the Arp2/3 complex. Specifically, both ArpC5 subunit isoforms, ArpC5 and ArpC5L, have been reported to fine-tune nucleation activity and branch junction stability. We have combined reverse genetics and cellular structural biology to describe how ArpC5 and ArpC5L differentially affect cell migration. Both define the structural stability of ArpC1 in branch junctions and, in turn, by determining protrusion characteristics, affect protein dynamics and actin network ultrastructure. ArpC5 isoforms also affect the positioning of members of the Ena/Vasodilator-stimulated phosphoprotein (VASP) family of actin filament elongators, which mediate ArpC5 isoform-specific effects on the actin assembly level. Our results suggest that ArpC5 and Ena/VASP proteins are part of a signaling pathway enhancing cell migration.
Topics: Actins; Actin-Related Protein 2-3 Complex; Microfilament Proteins; Actin Cytoskeleton; Protein Isoforms
PubMed: 36662867
DOI: 10.1126/sciadv.add6495 -
The Journal of Cell Biology Aug 2022IRSp53 (aka BAIAP2) is a scaffold protein that couples membranes with the cytoskeleton in actin-filled protrusions such as filopodia and lamellipodia. The protein is...
IRSp53 (aka BAIAP2) is a scaffold protein that couples membranes with the cytoskeleton in actin-filled protrusions such as filopodia and lamellipodia. The protein is abundantly expressed in excitatory synapses and is essential for synapse development and synaptic plasticity, although with poorly understood mechanisms. Here we show that specific multivalent interactions between IRSp53 and its binding partners PSD-95 or Shank3 drive phase separation of the complexes in solution. IRSp53 can be enriched to the reconstituted excitatory PSD (ePSD) condensates via bridging to the core and deeper layers of ePSD. Overexpression of a mutant defective in the IRSp53/PSD-95 interaction perturbs synaptic enrichment of IRSp53 in mouse cortical neurons. The reconstituted PSD condensates promote bundled actin filament formation both in solution and on membranes, via IRSp53-mediated actin binding and bundling. Overexpression of mutants that perturb IRSp53-actin interaction leads to defects in synaptic maturation of cortical neurons. Together, our studies provide potential mechanistic insights into the physiological roles of IRSp53 in synapse formation and function.
Topics: Actin Cytoskeleton; Actins; Animals; Mice; Microfilament Proteins; Nerve Tissue Proteins; Neuronal Plasticity; Neurons; Post-Synaptic Density; Pseudopodia; Synapses
PubMed: 35819332
DOI: 10.1083/jcb.202105035 -
Communications Biology Sep 2021The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the...
The proteins that make up the actin cytoskeleton can self-assemble into a variety of structures. In vitro experiments and coarse-grained simulations have shown that the actin crosslinking proteins α-actinin and fascin segregate into distinct domains in single actin bundles with a molecular size-dependent competition-based mechanism. Here, by encapsulating actin, α-actinin, and fascin in giant unilamellar vesicles (GUVs), we show that physical confinement can cause these proteins to form much more complex structures, including rings and asters at GUV peripheries and centers; the prevalence of different structures depends on GUV size. Strikingly, we found that α-actinin and fascin self-sort into separate domains in the aster structures with actin bundles whose apparent stiffness depends on the ratio of the relative concentrations of α-actinin and fascin. The observed boundary-imposed effect on protein sorting may be a general mechanism for creating emergent structures in biopolymer networks with multiple crosslinkers.
Topics: Actin Cytoskeleton; Actins; Carrier Proteins; Humans; Microfilament Proteins
PubMed: 34584211
DOI: 10.1038/s42003-021-02653-6 -
Immunity Jan 2019Defining pathways that differentially regulate graft-reactive versus anti-microbial T cell responses could facilitate more precise manipulation of immune responses in...
Defining pathways that differentially regulate graft-reactive versus anti-microbial T cell responses could facilitate more precise manipulation of immune responses in the context of organ and tissue transplantation. Here, Jayachandran et al. (2019) identify a coronin-1-PDE4-cAMP axis that, when perturbed, results in the induction of transplantation tolerance while maintaining anti-microbial immunity.
Topics: Immunity; Microfilament Proteins; Signal Transduction; T-Lymphocytes; Transplantation Tolerance
PubMed: 30650379
DOI: 10.1016/j.immuni.2018.12.030 -
Nature Structural & Molecular Biology Jun 2022Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis and other cellular processes by organizing strikingly different actin...
Plastins/fimbrins are conserved actin-bundling proteins contributing to motility, cytokinesis and other cellular processes by organizing strikingly different actin assemblies as in aligned bundles and branched networks. We propose that this ability of human plastins stems from an allosteric communication between their actin-binding domains (ABD1/2) engaged in a tight spatial association. Here we show that ABD2 can bind actin three orders of magnitude stronger than ABD1, unless the domains are involved in an equally strong inhibitory engagement. A mutation mimicking physiologically relevant phosphorylation at the ABD1-ABD2 interface greatly weakened their association, dramatically potentiating actin cross-linking. Cryo-EM reconstruction revealed the ABD1-actin interface and enabled modeling of the plastin bridge and domain separation in parallel bundles. We predict that a strong and tunable allosteric inhibition between the domains allows plastins to modulate the cross-linking strength, contributing to remodeling of actin assemblies of different morphologies defining the unique place of plastins in actin organization.
Topics: Actin Cytoskeleton; Actins; Allosteric Regulation; Humans; Membrane Glycoproteins; Microfilament Proteins
PubMed: 35589838
DOI: 10.1038/s41594-022-00771-1 -
The American Journal of the Medical... Mar 2020Fascin-1 and actinin-4 are involved in key processes of tumor cell adhesion, migration and metastasis. Actinin-4 plays an important role in promotion of cell...
BACKGROUND
Fascin-1 and actinin-4 are involved in key processes of tumor cell adhesion, migration and metastasis. Actinin-4 plays an important role in promotion of cell proliferation, whereas fascin-1 regulates cellular motility. Its over-expression leads to the loss of cell adhesion and metastasis. The aim of our study was to assess fascin-1 and actinin-4 expression in normal pancreatic ducts and in pancreatic intraepithelial neoplasia (PanIN) - precursor lesion of pancreatic ductal adenocarcinoma (PDAC).
MATERIALS AND METHODS
The study involved 70 patients treated surgically due to PDAC, cysts and pancreatitis, who had also been diagnosed with pancreatic intraepithelial neoplasia. Fascin-1 and actinin-4 expressions were evaluated using the immunohistochemistry method.
RESULTS
A statistically significant relationship was observed between the expression of fascin-1 and actinin-4 (cytoplasmic) and patients' age (P = 0.01, P = 0.002, respectively). The expression of fascin-1 and actinin-4 was associated with the diagnosis (P <0.001, P = 0.04, respectively). Statistical analysis revealed correlations of fascin-1 and actinin-4 expressions with the presence and grade of PanIN (P < 0.001, P = 0.002, respectively). The expression of these proteins was observed in each degree of PanIN and increased with the pancreatic intraepithelial neoplasia progression.
CONCLUSIONS
The expression of fascin-1 and actinin-4 is connected with the degree of PanIN advancement and depends on the type of the primary disease. Overexpression of these proteins may be linked to cytological and architectural abnormalities observed in advanced PanIN. Elevated expression of fascin-1 and actinin-4 indicates the role of these proteins in the progression from PanIN to PDAC.
Topics: Actinin; Aged; Carcinoma in Situ; Carcinoma, Pancreatic Ductal; Carrier Proteins; Disease Progression; Female; Humans; Male; Microfilament Proteins; Middle Aged; Pancreatic Neoplasms; Poland
PubMed: 31889512
DOI: 10.1016/j.amjms.2019.11.013 -
Sheng Li Xue Bao : [Acta Physiologica... Apr 2024There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers... (Review)
Review
There are three main classes of actin nucleation factors: Arp2/3 complexes, Spire and Formin. Spire assembles microfilaments by nucleating stable longitudinal tetramers and binding actin to the growing end of the microfilament. As early as 1999, Wellington et al. identified Spire as an actin nucleating agent, however, over the years, most studies have focused on Arp2/3 and Formin proteins; there has been relatively less research on Spire as a member of the actin nucleating factors. Recent studies have shown that Spire is involved in the vesicular transport through the synthesis of actin and plays an important role in neural development. In this paper, we reviewed the structure, expression and function of Spire, and its association with disease in order to identify meaningful potential directions for studies on Spire.
Topics: Microfilament Proteins; Humans; Animals; Actins; Actin-Related Protein 2-3 Complex; Actin Cytoskeleton; Nuclear Proteins
PubMed: 38658382
DOI: No ID Found -
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