Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells

(1) Background: The unusual accumulation of Na,K-ATPase complexes in the brush border membrane of choroid plexus epithelial cells have intrigued researchers for decades. However, the full range of the expressed Na,K-ATPase subunits and their relation to the microvillus cytoskeleton remains unknown. (2) Methods: RT-PCR analysis, co-immunoprecipitation, native PAGE, mass spectrometry, and differential centrifugation were combined with high-resolution immunofluorescence histochemistry, proximity ligase assays, and stimulated emission depletion (STED) microscopy on mouse choroid plexus cells or tissues in order to resolve these issues. (3) Results: The choroid plexus epithelium expresses Na,K-ATPase subunits α1, α2, β1, β2, β3, and phospholemman. The α1, α2, β1, and β2, subunits are all localized to the brush border membrane, where they appear to form a complex. The ATPase complexes may stabilize in the brush border membrane via anchoring to microvillar actin indirectly through ankyrin-3 or directly via other co-precipitated proteins. Aquaporin 1 (AQP1) may form part of the proposed multi-protein complexes in contrast to another membrane protein, the Na-K-2Cl cotransporter 1 (NKCC1). NKCC1 expression seems necessary for full brush border membrane accumulation of the Na,K-ATPase in the choroid plexus. (4) Conclusion: A multitude of Na,K-ATPase subunits form molecular complexes in the choroid plexus brush border, which may bind to the cytoskeleton by various alternative actin binding proteins.     

Myopathy-Sensitive G-Actin Segment 227-235 Is Involved in Salt-Induced Stabilization of Contacts within the Actin Filament

Formation of stable actin filaments, critically important for actin functions, is determined by the ionic strength of the solution. However, not much is known about the elements of the actin fold involved in ionic-strength-dependent filament stabilization. In this work, F-actin was destabilized by Cu²⁺ binding to Cys374, and the effects of solvent conditions on the dynamic properties of F-actin were correlated with the involvement of Segment 227-235 in filament stabilization. The results of our work show that the presence of Mg²⁺ at the high-affinity cation binding site of Cu-modified actin polymerized with MgCl₂ strongly enhances the rate of filament subunit exchange and promotes the filament instability. In the presence of 0.1 M KCl, the filament subunit exchange was 2–3-fold lower than that in the MgCl₂-polymerized F-actin. This effect correlates with the reduced accessibility of the D-loop and Segment 227-235 on opposite filament strands, consistent with an ionic-strength-dependent conformational change that modulates involvement of Segment 227-235 in stabilization of the intermonomer interface. KCl may restrict the mobility of the α-helix encompassing part of Segment 227-235 and/or be bound to Asp236 at the boundary of Segment 227-235. These results provide experimental evidence for the involvement of Segment 227-235 in salt-induced stabilization of contacts within the actin filament and suggest that they can be weakened by mutations characteristic of actin-associated myopathies.     

Melanin Distribution in Human Skin: Influence of Cytoskeletal,Polarity, and Centrosome-Related Machinery of Stratum basale Keratinocytes

Melanin granules cluster within supra-nuclear caps in basal keratinocytes (KCs) of the human epidermis, where they protect KC genomic DNA against ultraviolet radiation (UVR) damage. While much is known about melanogenesis in melanocytes (MCs) and a moderate amount about melanin transfer from MC to KC, we know little about the fate of melanin once inside KCs. We recently reported that melanin fate in progenitor KCs is regulated by rare asymmetric organelle movement during mitosis. Here, we explore the role of actin, microtubules, and centrosome-associated machinery in distributing melanin within KCs. Short-term cultures of human skin explants were treated with cytochalasin-B and nocodazole to target actin filaments and microtubules, respectively. Treatment effects on melanin distribution were assessed by the Warthin–Starry stain, on centrosome-associated proteins by immunofluorescence microscopy, and on co-localisation with melanin granules by brightfield microscopy. Cytochalasin-B treatment disassembled supra-nuclear melanin caps, while nocodazole treatment moved melanin from the apical to basal KC domain. Centrosome and centriolar satellite-associated proteins showed a high degree of co-localisation with melanin. Thus, once melanin granules are transferred to KCs, their preferred apical distribution appears to be facilitated by coordinated movement of centrosomes and centriolar satellites. This mechanism may control melanin’s strategic position within UVR-exposed KCs.     

Pathophysiological Roles of Actin-Binding Scaffold Protein,Ezrin

Ezrin is one of the members of the ezrin/radixin/moesin (ERM) family of proteins. It was originally discovered as an actin-binding protein in the microvilli structure about forty years ago. Since then, it has been revealed as a key protein with functions in a variety of fields including cell migration, survival, and signal transduction, as well as functioning as a structural component. Ezrin acts as a cross-linker of membrane proteins or phospholipids in the plasma membrane and the actin cytoskeleton. It also functions as a platform for signaling molecules at the cell surface. Moreover, ezrin is regarded as an important target protein in cancer diagnosis and therapy because it is a key protein involved in cancer progression and metastasis, and its high expression is linked to poor survival in many cancers. Small molecule inhibitors of ezrin have been developed and investigated as candidate molecules that suppress cancer metastasis. Here, we wish to comprehensively review the roles of ezrin from the pathophysiological points of view.     

Actin-Associated Proteins and Small Molecules Targeting the Actin Cytoskeleton

Actin-associated proteins (AAPs) act on monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin) to regulate their dynamics and architectures which ultimately control cell movement, shape change, division; organelle localization and trafficking. Actin-binding proteins (ABPs) are a subset of AAPs. Since actin was discovered as a myosin-activating protein (hence named actin) in 1942, the protein has also been found to be expressed in non-muscle cells, and numerous AAPs continue to be discovered. This review article lists all of the AAPs discovered so far while also allowing readers to sort the list based on the names, sizes, functions, related human diseases, and the dates of discovery. The list also contains links to the UniProt and Protein Atlas databases for accessing further, related details such as protein structures, associated proteins, subcellular localization, the expression levels in cells and tissues, mutations, and pathology. Because the actin cytoskeleton is involved in many pathological processes such as tumorigenesis, invasion, and developmental diseases, small molecules that target actin and AAPs which hold potential to treat these diseases are also listed.     

Aurora Kinase A Is Involved in Controlling the Localization of Aquaporin-2 in Renal Principal Cells

The cAMP-dependent aquaporin-2 (AQP2) redistribution from intracellular vesicles into the plasma membrane of renal collecting duct principal cells induces water reabsorption and fine-tunes body water homeostasis. However, the mechanisms controlling the localization of AQP2 are not understood in detail. Using immortalized mouse medullary collecting duct (MCD4) and primary rat inner medullary collecting duct (IMCD) cells as model systems, we here discovered a key regulatory role of Aurora kinase A (AURKA) in the control of AQP2. The AURKA-selective inhibitor Aurora-A inhibitor I and novel derivatives as well as a structurally different inhibitor, Alisertib, prevented the cAMP-induced redistribution of AQP2. Aurora-A inhibitor I led to a depolymerization of actin stress fibers, which serve as tracks for the translocation of AQP2-bearing vesicles to the plasma membrane. The phosphorylation of cofilin-1 (CFL1) inactivates the actin-depolymerizing function of CFL1. Aurora-A inhibitor I decreased the CFL1 phosphorylation, accounting for the removal of the actin stress fibers and the inhibition of the redistribution of AQP2. Surprisingly, Alisertib caused an increase in actin stress fibers and did not affect CFL1 phosphorylation, indicating that AURKA exerts its control over AQP2 through different mechanisms. An involvement of AURKA and CFL1 in the control of the localization of AQP2 was hitherto unknown.     

Coordination Environment of Cu(II) Ions Bound to N-Terminal Peptide Fragments of Angiogenin Protein

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH₂), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.     

豌豆肌动蛋白异型体PEAc3基因的克隆及生物信息学分析

根据NCBI文库中检索到的序列,采用RT-PCR技术克隆了豌豆肌动蛋白异型体PEAc3完整编码区的cD-NA序列,基因全长1134bp,开放阅读框编码377个氨基酸。氨基酸序列分析表明,该基因的编码蛋白PEAc3为可溶性蛋白,分子量41 668.7Da,没有信号肽部分,呈酸性,PEAc3包括ACTINS_1（编号：PS00406）、ACTINS_ACT_LIKE（编号：PS01132）以及ACTINS_2（编号：PS00432）3组肌动蛋白序列指纹图谱（Signature）。序列同源性分析表明,PEAc3与其它上百种植物肌动蛋白同源性高达89.07%。并讨论了模体中可结合配体的位点与功能之间的联系。     

Whole-mount preparations of mouse lens epithelium for the fluorescent cytological study of actin

A technique is given for the preparation of a sheet of epithelial cells from the capsule of the crystalline lens. A new method is described for fixation and staining with fluorescent phalloidin or actin antibody in order to localize the actin cytoskeleton in this tissue. Optical section of the preparation resolves such actin features as apical polygonal arrays, sequestered actin bundles, perinuclear actin aggregates, observed here for the first time, and filamentous networks in the basal region of the cell. This method is superior to previous ones in its ability to preserve actin-abundant sectors distinctively.     

Molecular Mechanisms of Host Cytoskeletal Rearrangements by Shigella Invasins

Pathogen-induced reorganization of the host cell cytoskeleton is a common strategy utilized in host cell invasion by many facultative intracellular bacteria, such as Shigella, Listeria, enteroinvasive E. coli and Salmonella. Shigella is an enteroinvasive intracellular pathogen that preferentially infects human epithelial cells and causes bacillary dysentery. Invasion of Shigella into intestinal epithelial cells requires extensive remodeling of the actin cytoskeleton with the aid of pathogenic effector proteins injected into the host cell by the activity of the type III secretion system. These so-called Shigella invasins, including IpaA, IpaC, IpgB1, IpgB2 and IpgD, modulate the actin-regulatory system in a concerted manner to guarantee efficient entry of the bacteria into host cells.