WHAMM is an Arp2/3 complex activator that binds microtubules and functions in ER to Golgi transport

The Arp2/3 complex is an actin nucleator that plays a critical role in many cellular processes. Its activities are regulated by nucleation-promoting factors (NPFs) that function primarily during plasma membrane dynamics. Here we identify a mammalian NPF called WHAMM (WASP homolog associated with actin, membranes, and microtubules) that localizes to the cis-Golgi apparatus and tubulo-vesicular membrane transport intermediates. The modular organization of WHAMM includes an N-terminal domain that mediates Golgi membrane association, a coiled-coil region that binds microtubules, and a WCA segment that stimulates Arp2/3-mediated actin polymerization. Overexpression and depletion studies indicate that WHAMM is important for maintaining Golgi structure and facilitating anterograde membrane transport. The ability of WHAMM to interact with microtubules plays a role in membrane tubulation, while its capacity to induce actin assembly promotes tubule elongation. Thus, WHAMM is an important regulator of membrane dynamics functioning at the interface of the microtubule and actin cytoskeletons.     

Rab1 recruits WHAMM during membrane remodeling but limits actin nucleation

Small G-proteins are key regulatory molecules that activate the actin nucleation machinery to drive cytoskeletal rearrangements during plasma membrane remodeling. However, the ability of small G-proteins to interact with nucleation factors on internal membranes to control trafficking processes has not been well characterized. Here we investigated roles for members of the Rho, Arf, and Rab G-protein families in regulating WASP homologue associated with actin, membranes, and microtubules (WHAMM), an activator of Arp2/3 complex–mediated actin nucleation. We found that Rab1 stimulated the formation and elongation of WHAMM-associated membrane tubules in cells. Active Rab1 recruited WHAMM to dynamic tubulovesicular structures in fibroblasts, and an active prenylated version of Rab1 bound directly to an N-terminal domain of WHAMM in vitro. In contrast to other G-protein–nucleation factor interactions, Rab1 binding inhibited WHAMM-mediated actin assembly. This ability of Rab1 to regulate WHAMM and the Arp2/3 complex represents a distinct strategy for membrane remodeling in which a Rab G-protein recruits the actin nucleation machinery but dampens its activity.     

Cytoskeletal regulation: coordinating actin and microtubule dynamics in membrane trafficking

The Arp2/3 complex is essential for actin nucleation and filament elongation in a variety of intracellular processes. This functional versatility is exerted through the regulation of its activity by nucleation-promoting factors (NPFs). The discovery of a new NPF, WHAMM, reveals unexpected connections between the actin and microtubule cytoskeletons and membrane dynamics during ER-to-Golgi transport.     

WASH, WHAMM and JMY: regulation of Arp2/3 complex and beyond

Arp2/3 complex mediates the nucleation of actin filaments in multiple subcellular processes, and is activated by nucleation-promoting factors (NPFs) from the Wiskott-Aldrich Syndrome family. In exciting new developments, this family has grown by three members: WASH, WHAMM and JMY, which extend the repertoire of dynamic membrane structures that are remodeled following Arp2/3 activation in vivo. These novel NPFs share an actin- and Arp2/3-interacting WCA module, and combine Arp2/3 activation with additional biochemical functions, including capping protein inhibition, microtubule engagement or Arp2/3-independent actin nucleation, none of which had been previously associated with canonical WCA-harboring proteins. Uncovering the physiological relevance of these unique activities will require concerted efforts from multiple disciplines, and is sure to impact our understanding of how the cytoskeleton controls so many dynamic subcellular events.     

WHAMM links actin assembly via the Arp2/3 complex to autophagy

Macroautophagy (hereafter autophagy) is the process by which cytosolic material destined for degradation is enclosed inside a double-membrane cisterna known as the autophagosome and processed for secretion and/or recycling. This process requires a large collection of proteins that converge on certain sites of the ER membrane to generate the autophagosome membrane. Recently, it was shown that actin accumulates around autophagosome precursors and could play a role in this process, but the mechanism and role of actin polymerization in autophagy were unknown. Here, we discuss our recent finding that the nucleation-promoting factor (NPF) WHAMM recruits and activates the Arp2/3 complex for actin assembly at sites of autophagosome formation on the ER. Using high-resolution, live-cell imaging, we showed that WHAMM forms dynamic puncta on the ER that comigrate with several autophagy markers, and propels the spiral movement of these puncta by an Arp2/3 complex-dependent actin comet tail mechanism. In starved cells, WHAMM accumulates at the interface between neighboring autophagosomes, whose number and size increases with WHAMM expression. Conversely, knocking down WHAMM, inhibiting the Arp2/3 complex or interfering with actin polymerization reduces the size and number of autophagosomes. These findings establish a link between Arp2/3 complex-mediated actin assembly and autophagy.     

The actin nucleation factors JMY and WHAMM enable a rapid Arp2/3 complex-mediated intrinsic pathway of apoptosis

The actin cytoskeleton is a well-known player in most vital cellular processes, but comparably little is understood about how the actin assembly machinery impacts programmed cell death pathways. In the current study, we explored roles for the human Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation factors in DNA damage-induced apoptosis. Inactivation of each WASP-family gene revealed that two of them, JMY and WHAMM, are necessary for rapid apoptotic responses. JMY and WHAMM participate in a p53-dependent cell death pathway by enhancing mitochondrial permeabilization, initiator caspase cleavage, and executioner caspase activation. JMY-mediated apoptosis requires actin nucleation via the Arp2/3 complex, and actin filaments are assembled in cytoplasmic territories containing clusters of cytochrome c and active caspase-3. The loss of JMY additionally results in significant changes in gene expression, including upregulation of the WHAMM-interacting G-protein RhoD. Depletion or deletion of RHOD increases cell death, suggesting that RhoD normally contributes to cell survival. These results give rise to a model in which JMY and WHAMM promote intrinsic cell death responses that can be opposed by RhoD.     

WHAMM is required for meiotic spindle migration and asymmetric cytokinesis in mouse oocytes

WASP homolog associated with actin, membranes and microtubules (WHAMM) is a newly discovered nucleation-promoting factor that links actin and microtubule cytoskeleton and regulates transport from the endoplasmic reticulum to the Golgi apparatus. However, knowledge of WHAMM is limited to interphase somatic cells. In this study, we examined its localization and function in mouse oocytes during meiosis. Immunostaining showed that in the germinal vesicle (GV) stage, there was no WHAMM signal; after meiosis resumption, WHAMM was associated with the spindle at prometaphase I (Pro MI), metaphase I (MI), telophase I (TI) and metaphase II (MII) stages. Nocodazole and taxol treatments showed that WHAMM was localized around the MI spindle. Depletion of WHAMM by microinjection of specific short interfering (si)RNA into the oocyte cytoplasm resulted in failure of spindle migration, disruption of asymmetric cytokinesis and a decrease in the first polar body extrusion rate during meiotic maturation. Moreover, actin cap formation was also disrupted after WHAMM depletion, confirming the failure of spindle migration. Taken together, our data suggest that WHAMM is required for peripheral spindle migration and asymmetric cytokinesis during mouse oocyte maturation.     

RhoD regulates cytoskeletal dynamics via the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules

The Rho GTPases have mainly been studied in association with their roles in the regulation of actin filament organization. These studies have shown that the Rho GTPases are essential for basic cellular processes, such as cell migration, contraction, and division. In this paper, we report that RhoD has a role in the organization of actin dynamics that is distinct from the roles of the better-studied Rho members Cdc42, RhoA, and Rac1. We found that RhoD binds the actin nucleation–promoting factor WASp homologue associated with actin Golgi membranes and microtubules (WHAMM), as well as the related filamin A–binding protein FILIP1. Of these two RhoD-binding proteins, WHAMM was found to bind to the Arp2/3 complex, while FILIP1 bound filamin A. WHAMM was found to act downstream of RhoD in regulating cytoskeletal dynamics. In addition, cells treated with small interfering RNAs for RhoD and WHAMM showed increased cell attachment and decreased cell migration. These major effects on cytoskeletal dynamics indicate that RhoD and its effectors control vital cytoskeleton-driven cellular processes. In agreement with this notion, our data suggest that RhoD coordinates Arp2/3-dependent and FLNa-dependent mechanisms to control the actin filament system, cell adhesion, and cell migration.     

Microtubule anchoring by cortical actin bundles prevents streaming of the oocyte cytoplasm

The localisation of the determinants of the body axis during Drosophila oogenesis is dependent on the microtubule (MT) cytoskeleton. Mutations in the actin binding proteins Profilin, Cappuccino (Capu) and Spire result in premature streaming of the cytoplasm and a reorganisation of the oocyte MT network. As a consequence, the localisation of axis determinants is abolished in these mutants. It is unclear how actin regulates the organisation of the MTs, or what the spatial relationship between these two cytoskeletal elements is. Here, we report a careful analysis of the oocyte cytoskeleton. We identify thick actin bundles at the oocyte cortex, in which the minus ends of the MTs are embedded. Disruption of these bundles results in cortical release of the MT minus ends, and premature onset of cytoplasmic streaming. Thus, our data indicate that the actin bundles anchor the MTs minus ends at the oocyte cortex, and thereby prevent streaming of the cytoplasm. We further show that actin bundle formation requires Profilin but not Capu and Spire. Thus, our results support a model in which Profilin acts in actin bundle nucleation, while Capu and Spire link the bundles to MTs. Finally, our data indicate how cytoplasmic streaming contributes to the reorganisation of the MT cytoskeleton. We show that the release of the MT minus ends from the cortex occurs independently of streaming, while the formation of MT bundles is streaming dependent.     

Under lock and key: Spatiotemporal regulation of WASP family proteins coordinates separate dynamic cellular processes

WASP family proteins are nucleation promoting factors that bind to and activate the Arp2/3 complex in order to stimulate nucleation of branched actin filaments. The WASP family consists of WASP, N-WASP, WAVE1-3, WASH, and the novel family members WHAMM and JMY. Each of the family members contains a C-terminus responsible for their nucleation promoting activity and unique N-termini that allow for them to be regulated in a spatiotemporal manner. Upon activation they reorganize the cytoskeleton for different cellular functions depending on their subcellular localization and regulatory protein interactions. Emerging evidence indicates that WASH, WHAMM, and JMY have functions that require the coordination of both actin polymerization and microtubule dynamics. Here, we review the mechanisms of regulation for each family member and their associated in vivo functions including cell migration, vesicle trafficking, and neuronal development.     

Activation of nucleation promoting factors for directional actin filament elongation: Allosteric regulation and multimerization on the membrane

Nucleation promoting factors (NPFs) activate the Arp2/3 complex to produce branched actin filaments. Branched actin filaments are observed in most organelles, and specific NPFs, such as WASP, N-WASP, WAVEs, WASH, and WHAMM, exist for each organelle. Interestingly, Arp2/3 and NPFs are both inactive by themselves, and thus require activation. The exposure of the Arp2/3 activating region, the VCA fragment, is recognized to be a key event in the activation of the NPFs. Together, small GTPase binding, phosphorylation, SH3 binding, and membrane binding promote VCA exposure synergistically. The increase in the local concentration of NPF by multimerization is thought to occur with the combination of such activators, to maximally activate the NPF and confine the region of actin polymerization. The mechanism of uni-directional filament extension beneath the membrane also is discussed.     

The direction of actin polymerization for vesicle fission suggested from membranes tubulated by the EFC/F-BAR domain protein FBP17

Actin polymerization mediated by the Arp2/3 complex is essential for membrane tubulation, vesicle formation and fission during clathrin-dependent endocytosis. However, the mechanism by which the polymerizing actin filaments participate in vesicle formation and fission has remained unclear. Our analyses revealed that actin polymerization occurs toward FBP17-induced membrane tubules, which are considered to be generated during endocytic vesicle formation. The tubulated membrane between the future endocytic vesicle and the plasma membrane is proposed to form an arc upon scission of the endocytic vesicle. Therefore, the actin polymerization toward the tubulated membrane may be gradually converted to those toward both the vesicles and the plasma membrane.     

Caldesmon inhibits Arp2/3-mediated actin nucleation

The Arp2/3 complex greatly accelerates actin polymerization, which is thought to play a major role in cell motility by inducing membrane protrusions including ruffling movements. Membrane ruffles contain a variety of actin-binding proteins, which would modulate Arp2/3-dependent actin polymerization. However, their exact roles in actin polymerization remain to be established. Because caldesmon is present in membrane ruffles, as well as in stress fibers, it may alter Arp2/3-mediated actin polymerization. We have found that caldesmon greatly retards Arp2/3-induced actin polymerization. Kinetic analyses have revealed that caldesmon inhibits the nucleation process, whereas it does not largely reduce elongation. Caldesmon is found to inhibit binding of Arp2/3 to F-actin, which apparently reduces the ability of F-actin as a secondary activator of Arp2/3-mediated nucleation. We also have found that the inhibition of the binding between actin and caldesmon either by Ca(2+)/calmodulin or by phosphorylation with cdc2 kinase reverses the inhibitory effect of caldesmon on Arp2/3-induced actin polymerization. Our results suggest that caldesmon may be a key protein that modulates membrane ruffling and that this may involve changes in caldesmon phosphorylation and/or intracellular calcium concentrations during signal transduction.     

Endophilin BAR domain drives membrane curvature by two newly identified structure-based mechanisms

The crescent-shaped BAR (Bin/Amphiphysin/Rvs-homology) domain dimer is a versatile protein module that senses and generates positive membrane curvature. The BAR domain dimer of human endophilin-A1, solved at 3.1 A, has a unique structure consisting of a pair of helix-loop appendages sprouting out from the crescent. The appendage's short helices form a hydrophobic ridge, which runs across the concave surface at its center. Examining liposome binding and tubulation in vitro using purified BAR domain and its mutants indicated that the ridge penetrates into the membrane bilayer and enhances liposome tubulation. BAR domain-expressing cells exhibited marked plasma membrane tubulation in vivo. Furthermore, a swinging-arm mutant lost liposome tubulation activity yet retaining liposome binding. These data suggested that the rigid crescent dimer shape is crucial for the tubulation. We here propose that the BAR domain drives membrane curvature by coordinate action of the crescent's scaffold mechanism and the ridge's membrane insertion in addition to membrane binding via amino-terminal amphipathic helix.     

Adenomatous polyposis coli protein nucleates actin assembly and synergizes with the formin mDia1

The tumor suppressor protein adenomatous polyposis coli (APC) regulates cell protrusion and cell migration, processes that require the coordinated regulation of actin and microtubule dynamics. APC localizes in vivo to microtubule plus ends and actin-rich cortical protrusions, and has well-documented direct effects on microtubule dynamics. However, its potential effects on actin dynamics have remained elusive. Here, we show that the C-terminal “basic” domain of APC (APC-B) potently nucleates the formation of actin filaments in vitro and stimulates actin assembly in cells. Nucleation is achieved by a mechanism involving APC-B dimerization and recruitment of multiple actin monomers. Further, APC-B nucleation activity is synergistic with its in vivo binding partner, the formin mDia1. Together, APC-B and mDia1 overcome a dual cellular barrier to actin assembly imposed by profilin and capping protein. These observations define a new function for APC and support an emerging view of collaboration between distinct actin assembly–promoting factors with complementary activities.     

An ESCRT–spastin interaction promotes fission of recycling tubules from the endosome

Mechanisms coordinating endosomal degradation and recycling are poorly understood, as are the cellular roles of microtubule (MT) severing. We show that cells lacking the MT-severing protein spastin had increased tubulation of and defective receptor sorting through endosomal tubular recycling compartments. Spastin required the ability to sever MTs and to interact with ESCRT-III (a complex controlling cargo degradation) proteins to regulate endosomal tubulation. Cells lacking IST1 (increased sodium tolerance 1), an endosomal sorting complex required for transport (ESCRT) component to which spastin binds, also had increased endosomal tubulation. Our results suggest that inclusion of IST1 into the ESCRT complex allows recruitment of spastin to promote fission of recycling tubules from the endosome. Thus, we reveal a novel cellular role for MT severing and identify a mechanism by which endosomal recycling can be coordinated with the degradative machinery. Spastin is mutated in the axonopathy hereditary spastic paraplegia. Zebrafish spinal motor axons depleted of spastin or IST1 also had abnormal endosomal tubulation, so we propose this phenotype is important for axonal degeneration.     

Actin dynamics at intracellular membranes: the Spir/formin nucleator complex

The assembly of actin monomers into filaments is a highly regulated basic cellular function. The structural organization of a cell, morphological changes or cell motility is dependent on actin filament dynamics. While within the last decade substantial knowledge has been acquired about actin dynamics at the cell membrane, today only little is known about the actin cytoskeleton and its functions at intracellular endosomal and organelle membranes. The Spir actin nucleators are specifically targeted towards endosomal membranes by a FYVE zinc finger membrane localization domain, and provide an important link to study the role of actin dynamics in the regulation of intracellular membrane transport. Spir proteins are the founding members of a novel class of actin nucleation factors, which initiate actin polymerization by binding of actin monomers to one or multiple Wiskott-Aldrich syndrome protein (WASp) homology 2 (WH2) domains. Although Spir proteins can nucleate actin polymerization in vitro by themselves, they form a regulatory complex with the distinct actin nucleators of the formin subgroup (Fmn) of formins. A cooperative mechanism in actin nucleation has been proposed. Ongoing studies on the function and regulation of the Spir proteins in vesicle transport processes will reveal important insights into actin dynamics at intracellular membranes and how this regulates the highly directed and controlled routes of intracellular membrane trafficking.     

The formin mDia2 stabilizes microtubules independently of its actin nucleation activity

A critical microtubule (MT) polarization event in cell migration is the Rho/mDia-dependent stabilization of a subset of MTs oriented toward the direction of migration. Although mDia nucleates actin filaments, it is unclear whether this or a separate activity of mDia underlies MT stabilization. We generated two actin mutants (K853A and I704A) in a constitutively active version of mDia2 containing formin homology domains 1 and 2 (FH1FH2) and found that they still induced stable MTs and bound to the MT TIP proteins EB1 and APC, which have also been implicated in MT stabilization. A dimerization-impaired mutant of mDia2 (W630A) also generated stable MTs in cells. We examined whether FH1FH2mDia2 had direct activity on MTs in vitro and found that it bound directly to MTs, stabilized MTs against cold- and dilution-induced disassembly, and reduced the rates of growth and shortening during MT assembly and disassembly, respectively. These results indicate that mDia2 has a novel MT stabilization activity that is separate from its actin nucleation activity.     

Influence of calmodulin on the human red cell membrane skeleton

The calcium receptor calmodulin interacts with components of the human red cell membrane skeleton as well as with the membrane. Under physiological salt conditions, calmodulin has a calcium-dependent affinity for spectrin, one of the major components of the membrane skeleton. It is apparent from our results that calmodulin inhibits the ability of erythrocyte spectrin (when preincubated with filamentous actin) to create nucleation centers and thereby to seed actin polymerization. The gelation of filamentous actin induced by spectrin tetramers is also inhibited by calmodulin. The inhibition is calcium dependent and decreases with increasing pH, similar to the binding of calmodulin to spectrin. Direct binding studies using aqueous two-phase partition indicate that calmodulin interferes with the binding of actin to spectrin. Even in the presence of protein 4.1, which is believed to stabilize the ternary complex, calmodulin has an inhibitory effect. Since calmodulin also inhibits the corresponding activities of brain spectrin (fodrin), it appears likely that calmodulin may modulate the organization of cytoskeletons containing actin and spectrin or spectrin analogues.     

Dynamin A, Myosin IB and Abp1 couple phagosome maturation to F-actin binding

The role of actin, class I myosins and dynamin in endocytic uptake processes is well characterized, but their role during endo-phagosomal membrane trafficking and maturation is less clear. In Dictyostelium, knockout of myosin IB (myoB) leads to a defect in membrane protein recycling from endosomes back to the plasma membrane. Here, we show that actin plays a central role in the morphology and function of the endocytic pathway. Indeed, latrunculin B (LatB) induces endosome tubulation, a phenotype also observed in dynamin A (dymA)-null cells. Knockout of dymA impairs phagosome acidification, whereas knockout of myoB delays reneutralization, a phenotype mimicked by a low dose of LatB. As a read out for actin-dependent processes during maturation, we monitored the capacity of purified phagosomes to bind F-actin in vitro, and correlated this with the presence of actin-binding and membrane-trafficking proteins. Phagosomes isolated from myoB-null cells showed an increased binding to F-actin, especially late phagosomes. In contrast, early phagosomes from dymA-null cells showed reduced binding to F-actin while late phagosomes were unaffected. We provide evidence that Abp1 is the main F-actin-binding protein in this assay and is central for the interplay between DymA and MyoB during phagosome maturation.