Philip G. Allen

Mutations in ACTN4, encoding α-actinin-4, cause familial focal segmental glomerulosclerosis

Focal and segmental glomerulosclerosis (FSGS) is a common, non-specific renal lesion. Although it is often secondary to other disorders, including HIV infection, obesity, hypertension and diabetes, FSGS also appears as an isolated, idiopathic condition. FSGS is characterized by increased urinary protein excretion and decreasing kidney function. Often, renal insufficiency in affected patients progresses to end-stage renal failure, a highly morbid state requiring either dialysis therapy or kidney transplantation. Here we present evidence implicating mutations in the gene encoding α-actinin-4 (ACTN4; ref. 2), an actin-filament crosslinking protein, as the cause of disease in three families with an autosomal dominant form of FSGS. In vitro, mutant α-actinin-4 binds filamentous actin (F-actin) more strongly than does wild-type α-actinin-4. Regulation of the actin cytoskeleton of glomerular podocytes may be altered in this group of patients. Our results have implications for understanding the role of the cytoskeleton in the pathophysiology of kidney disease and may lead to a better understanding of the genetic basis of susceptibility to kidney damage.

Visualization of retroviral replication in living cells reveals budding into multivesicular bodies.

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host‐derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)‐infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus‐like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome‐based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.

CHEMOTACTIC SIGNALING PATHWAYS IN NEUTROPHILS: FROM RECEPTOR TO ACTIN ASSEMBLY

In this review, we present an overview of the signaling elements between neutrophil chemotactic receptors and the actin cytoskeleton that drives cell motility. From receptor-ligand interactions, activation of heterotrimeric G-proteins, their downstream effectors PLC and PI-3 kinase, the activation of small GTPases of the Rho family, and their regulation of particular cytoskeletal regulatory proteins, we describe pathways specific to the chemotaxing neutrophil and elements documented to be important for neutrophil function.

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.

VASP Protects Actin Filaments From Gelsolin: An In Vitro Study With Implications for Platelet Actin Reorganizations

An initial step in platelet shape change is disassembly of actin filaments, which are then reorganized into new actin structures, including filopodia and lamellipodia. This disassembly is thought to be mediated primarily by gelsolin, an abundant actin filament-severing protein in platelets. Shape change is inhibited by VASP, another abundant actin-binding protein. Paradoxically, in vitro VASP enhances formation of actin filaments and bundles them, activities that would be expected to increase shape change, not inhibit it. We hypothesized that VASP might inhibit shape change by stabilizing filaments and preventing their disassembly by gelsolin. Such activity would explain VASPs known physiological role. Here, we test this hypothesis in vitro using either purified recombinant or endogenous platelet VASP by fluorescence microscopy and biochemical assays. VASP inhibited gelsolins ability to disassemble actin filaments in a dose-dependent fashion. Inhibition was detectable at the low VASP:actin ratio found inside the platelet (1:40 VASP:actin). Gelsolin bound to VASP-actin filaments at least as well as to actin alone. VASP inhibited gelsolin-induced nucleation at higher concentrations (1:5 VASP:actin ratios). VASPs affinity for actin (K(d) approximately 0.07 microM) and its ability to promote polymerization (1:20 VASP actin ratio) were greater with Ca(++)-actin than with Mg(++)-actin (K(d) approximately 1 microM and 1:1 VASP), regardless of the presence of gelsolin. By immunofluorescence, VASP and gelsolin co-localized in the filopodia and lamellipodia of platelets spreading on glass, suggesting that these in vitro interactions could take place within the cell as well. We conclude that VASP stabilizes actin filaments to the severing effects of gelsolin but does not inhibit gelsolin from binding to the filaments. These results suggest a new concept for actin dynamics inside cells: that bundling proteins protect the actin superstructure from disassembly by severing, thereby preserving the integrity of the cytoskeleton.

C-terminal variations in ?-thymosin family members specify functional differences in actin-binding properties

Mammalian cells express several isoforms of β‐thymosin, a major actin monomer sequestering factor, including thymosins β4, β10, and β15. Differences in actin‐binding properties of different β‐thymosin family members have not been investigated. We find that thymosin β15 binds actin with a 2.4‐fold higher affinity than does thymosin β4. Mutational analysis was performed to determine the amino acid differences in thymosin β15 that specify its increased actin‐affinity. Previous work with thymosin β4 identified an α‐helical domain, as well as a conserved central motif, as crucial for actin binding. Mutational analysis confirms that these domains are also vital for actin binding in thymosin β15, but that differences in these domains are not responsible for the variation in actin‐binding properties between thymosins β4 and β15. Truncation of the unique C‐terminal residues in thymosin β15 inhibits actin binding, suggesting that this domain also has an important role in mediating actin‐binding affinity. Replacement of the 10 C‐terminal amino acids of thymosin β15 with those of thymosin β4 did, however, reduce the actin‐binding affinity of the hybrid relative to thymosin β15. Similarly, replacement of the thymosin β4 C‐terminal amino acids with those of thymosin β15 led to increased actin binding. We conclude that functional differences between closely related β‐thymosin family members are, in part, specified by the C‐terminal variability between these isoforms. Such differences may have consequences for situations where β‐thymosins are differentially expressed as in embryonic development and in cancer. J. Cell. Biochem. 77:277–287, 2000.

Dynamics of filamentous actin organization in the sea urchin egg cortex during early cleavage divisions: implications for the mechanism of cytokinesis.

We have used confocal laser scanning microscopy in conjunction with BODIPY-phallacidin staining of filamentous actin to investigate changes in the quantity and organization of cortical actin during the first two cell cycles following fertilization in eggs of the sea urchin Strongylocentrotus purpuratus. Quantification of fluorescent phallacidin staining reveals that the amount of filamentous actin (F-actin) in the cortex undergoes cyclical increases and decreases during early cleavage divisions, peaking near the beginning of the cell cycle and decreasing to a minimum at cytokinesis. Changes in the content of cortical F-actin are accompanied by the growth and disappearance of rootlet-like bundles of actin filaments which extend from the bases of microvilli that cover the surface of the egg. Actin rootlets reach their maximum degree of development by 20 min postfertilization, and then gradually decrease in number and length over the next 40 min. Small actin rootlets persist until cleavage, disappear during cytokinesis, and reform following division. The formation of actin rootlets requires cytoplasmic alkalization and is inhibited by cytochalasin D. Cytochalasin D washout experiments demonstrate that assembly of the cortical actin cytoskeleton can be blocked until 5 min before the onset of cleavage and still allow normal cytokinesis. These results illustrate the dynamic nature of cortical actin organization during early development and demonstrate that cytokinesis occurs at the point of minimum cortical F-actin content. They further demonstrate that cytokinesis can occur in embryos in which the normal developmental sequence of changes in cortical actin organization has been blocked by treatment with cytochalasin D, suggesting that these changes do not function in the establishment of the contractile apparatus for cytokinesis, but rather serve other developmental functions. Cell Motil. Cytoskeleton 36:30-42, 1997.

Deconstructing gelsolin: identifying sites that mimic or alter binding to actin and phosphoinositides

Gelsolin is involved in cytoskeletal remodeling as it can fragment and guide reassembly of actin networks. Recent advances in defining the structure of gelsolin identified functionally important sites. These structural insights could lead to the design of small molecule analogs to enhance, inhibit or mimic the functions of gelsolin.

Brains and brawn: plectin as regulator and reinforcer of the cytoskeleton.

Plectin is a 580 kDa intracellular protein, previously shown to link intermediate filaments with microtubules, actin filaments, and membrane components. Disruption of the plectin gene in humans and in mice results in severe skin blistering and muscular degeneration, consistent with plectins structural role in stabilizing cells against mechanical force. However, recent work by Andra et al. characterizing cells from plectin-deficient mice demonstrates that in addition to this structural role, plectin also modulates the dynamics of the actin cytoskeleton. This makes plectin unusual in that it serves both to reinforce and crosslink intermediate filament attachments to membranes and other cytoskeletal polymers and to regulate actin dynamics in cells.

Functional consequences of disulfide bond formation in gelsolin

Gelsolin is an actin monomer binding and filament severing protein synthesized in plasma and cytoplasmic forms differing by an N‐terminal amino acid extension and a disulfide bond between Cys‐188 and Cys‐201. To determine whether this bond altered gelsolin regulation or function, oxidized and reduced plasma gelsolins were assayed for severing, monomer binding and nucleation activity at a variety of rate‐limiting calcium concentrations. The results indicate that the disulfide bond in domain 2 of gelsolin influences the transmission of information from C‐terminal regulatory sites to functional sites in the N‐terminus.