Thomas P. Stossel

Filamins as integrators of cell mechanics and signalling

Filamins are large actin-binding proteins that stabilize delicate three-dimensional actin webs and link them to cellular membranes. They integrate cellular architectural and signalling functions and are essential for fetal development and cell locomotion. Here, we describe the history, structure and function of this group of proteins.

Thrombin receptor ligation and activated rac uncap actin filament barbed ends through phosphoinositide synthesis in permeabilized human platelets

Cells respond to diverse external stimuli by polymerizing cytoplasmic actin, and recent evidence indicates that GTPases can specify where this polymerization takes place. Actin assembly in stimulated blood platelets occurs where sequestered monomers add onto the fast-growing (barbed) ends of actin filaments (F-actin), which are capped in the resting cells. We report that D3 and D4 polyphosphoinositides, Pl(4)P, Pl(4,5)P2, Pl(3,4)P2, and Pl(3,4,5)P3, uncap F-actin in resting permeabilized platelets. The thrombin receptor-activating peptide (TRAP), GTP, and GTP gamma S, but not GDP beta S, also uncap F-actin in permeabilized platelets. GDP beta S inhibits TRAP-induced F-actin uncapping, and Pl(4,5)P2 overcomes this inhibition. Constitutively active mutant Rac, but not Rho, activates uncapping of F-actin. Pl(4,5)P2-binding peptides derived from gelsolin inhibit F-actin uncapping by TRAP, Rac, and GTP gamma S. TRAP and Rac induce rapid Pl(4,5)P2 synthesis in permeabilized platelets. The findings establish a signaling pathway for actin assembly involving Rac in which the final message is phosphoinositide-mediated F-actin uncapping.

Control of cytoplasmic actin gel-sol transformation by gelsolin, a calcium-dependent regulatory protein.

The peripheral cytoplasm of macrophages is involved in the control of locomotion, secretion and endocytosis, events common to many eukaryotic cells. During these activities, the cortical cytoplasm, which contains numerous actin filaments1,2, appears to undergo reversible gel–sol transformations3: cycles of gelation and solation are demonstrable in suitably prepared macrophage extracts, and the gels contain tangled actin filaments4. These changes in consistency of cytoplasmic actin may regulate motile events in the macrophage periphery. Calcium in micromolar concentrations prevents gelation of crude macrophage cytoplasmic extracts4, providing a possible link to abundant indirect evidence implicating calcium in the regulation of locomotion, secretion and endocytosis5. Similar calcium-sensitive gelation phenomena occur in crude cell extracts from diverse cell types and may have a relevance for control of cell movements in general6–11. Actin gelation results from the cross-linking of actin filaments (F-actin) by other proteins. In macrophages, a high molecular weight actin-binding protein (ABP) is the principal actin cross-linking protein12. Cross-linking of actin by these purified actin-binding proteins, however, is insensitive to changes in the calcium concentration4,12, so that another factor must mediate the expression of a calcium effect. We have now isolated such a calcium-dependent regulatory protein from macrophages and call it gelsolin.

Hemostatic, inflammatory, and fibroblast responses are blunted in mice lacking gelsolin

Gelsolin, an 82 kDa actin-binding protein, has potent actin filament-severing activity in vitro. To investigate the in vivo function of gelsolin, transgenic gelsolin-null (Gsn-) mice were generated and found to have normal embryonic development and longevity. However, platelet shape changes are decreased in Gsn- mice, causing prolonged bleeding times. Neutrophil migration in vivo into peritoneal exudates and in vitro is delayed. Gsn- dermal fibroblasts have excessive actin stress fibers and migrate more slowly than wild-type fibroblasts, but have increased contractility in vitro. These observations establish the requirement of gelsolin for rapid motile responses in cell types involved in stress responses such as hemostasis, inflammation, and wound healing. Neither gelsolin nor other proteins with similar actin filament-severing activity are expressed in early embryonic cells, indicating that this mechanism of actin filament dynamics is not essential for motility during early embryogenesis.

Endocytic protein intersectin-l regulates actin assembly via Cdc42 and N-WASP

Intersectin-s is a modular scaffolding protein regulating the formation of clathrin-coated vesicles. In addition to the Eps15 homology (EH) and Src homology 3 (SH3) domains of intersectin-s, the neuronal variant (intersectin-l) also has Dbl homology (DH), pleckstrin homology (PH) and C2 domains. We now show that intersectin-l functions through its DH domain as a guanine nucleotide exchange factor (GEF) for Cdc42. In cultured cells, expression of DH-domain-containing constructs cause actin rearrangements specific for Cdc42 activation. Moreover, in vivo studies reveal that stimulation of Cdc42 by intersectin-l accelerates actin assembly via N-WASP and the Arp2/3 complex. N-WASP binds directly to intersectin-l and upregulates its GEF activity, thereby generating GTP-bound Cdc42, a critical activator of N-WASP. These studies reveal a role for intersectin-l in a novel mechanism of N-WASP activation and in regulation of the actin cytoskeleton.

The Clearance Mechanism of Chilled Blood Platelets

Platelet transfusion is a very common lifesaving medical procedure. Not widely known is the fact that platelets, unlike other blood cells, rapidly leave the circulation if refrigerated prior to transfusion. This peculiarity requires blood services to store platelets at room temperature, limiting platelet supplies for clinical needs. Here, we describe the mechanism of this clearance system, a longstanding mystery. Chilling platelets clusters their von Willebrand (vWf) receptors, eliciting recognition of mouse and human platelets by hepatic macrophage complement type 3 (CR3) receptors. CR3-expressing but not CR3-deficient mice exposed to cold rapidly decrease platelet counts. Cooling primes platelets for activation. We propose that platelets are thermosensors, primed at peripheral sites where most injuries occurred throughout evolution. Clearance prevents pathologic thrombosis by primed platelets. Chilled platelets bind vWf and function normally in vitro and ex vivo after transfusion into CR3-deficient mice. Therefore, GPIb modification might permit cold platelet storage.

FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodelling

FilGAP is a newly recognized filamin A (FLNa)-binding RhoGTPase-activating protein. The GTPase-activating protein (GAP) activity of FilGAP is specific for Rac and FLNa binding targets FilGAP to sites of membrane protrusion, where it antagonizes Rac in vivo. Dominant-negative FilGAP constructs lacking GAP activity or knockdown of endogenous FilGAP by small interference RNA (siRNA) induce spontaneous lamellae formation and stimulate cell spreading on fibronectin. Knockdown of endogenous FilGAP abrogates ROCK-dependent suppression of lamellae. Conversely, forced expression of FilGAP induces numerous blebs around the cell periphery and a ROCK-specific inhibitor suppresses bleb formation. ROCK phosphorylates FilGAP, and this phosphorylation stimulates its RacGAP activity and is a requirement for FilGAP-mediated bleb formation. FilGAP is, therefore, a mediator of the well-established antagonism of Rac by RhoA that suppresses leading edge protrusion and promotes cell retraction to achieve cellular polarity.

Filamin is essential in actin cytoskeletal assembly mediated by p21-activated kinase 1.

The serine/threonine kinase p21-activated kinase 1 (Pak1) controls the actin cytoskeletal and ruffle formation through mechanisms that are independent of GTPase activity. Here we identify filamin FLNa as a Pak1-interacting protein through a yeast two-hybrid screen using the amino terminus of Pak1 as a bait. FLNa is stimulated by physiological signalling molecules to undergo phosphorylation by Pak1 and to interact and colocalize with endogenous Pak1 in membrane ruffles. The ruffle-forming activity of Pak1 is functional in FLNa-expressing cells but not in FLNa-deficient cells. In FLNa, the Pak1-binding site involves tandem repeat 23 in the carboxyl terminus and phosphorylation takes place on serine 2152. The FLNa-binding site in Pak1 is localized between amino acids 52 and 132 in the conserved Cdc42/Rac-interacting (CRIB) domain; accordingly, FLNa binding to the CRIB domain stimulates Pak1 kinase activity. Our results indicate that FLNa may be essential for Pak1-induced cytoskeletal reorganization and that the two-way regulatory interaction between Pak1 and FLNa may contribute to the local stimulation of Pak1 activity and its targets in cytoskeletal structures.

Gelsolin is a downstream effector of rac for fibroblast motility

Rac, a member of the rho family of GTPases, when activated transmits signals leading to actin‐based membrane ruffling in fibroblasts. Compared with wild‐type fibroblasts, gelsolin null (Gsn−) dermal fibroblasts have a markedly reduced ruffling response to serum or EGF stimulation, which signal through rac. Bradykinin‐induced filopodial formation, attributable to activation of cdc42, is similar in both cell types. Wild‐type fibroblasts exhibit typical lamellipodial extension during translational locomotion, whereas Gsn− cells move 50% slower using structures resembling filopodia. Multiple Gsn− tissues as well as Gsn− fibroblasts overexpress rac, but not cdc42 or rho, 5‐fold. Re‐expression of gelsolin in Gsn− fibroblasts by stable transfection or adenovirus reverts the ruffling response, translational motility and rac expression to normal. Rac migrates to the cell membrane following EGF stimulation in both cell types. Gelsolin is an essential effector of rac‐mediated actin dynamics, acting downstream of rac recruitment to the membrane.

The filamins Organizers of cell structure and function

Filamin A (FLNa), the first non-muscle actin filament cross-linking protein, was identified in 1975. Thirty five years of FLNa research has revealed its structure in great detail, discovered its isoforms (FLNb and c), and identified over 90 binding partners including channels, receptors, intracellular signaling molecules, and even transcription factors. Due to this diversity, mutations in human FLN genes result in a wide range of anomalies with moderate to lethal consequences. This review focuses on the structure and functions of FLNa in cell migration and adhesion.