JMY is involved in anterograde vesicle trafficking from the trans-Golgi network.

Junction-mediating and regulatory protein (JMY) was originally identified as a transcriptional co-factor in the p53-response to DNA damage. Aside from this nuclear function, recent years have uncovered an additional function of JMY, namely in cytoskeleton remodelling and actin assembly. The C-terminus of JMY comprises a canonical VCA-module, the sequence signature of Arp2/3 complex activators. Furthermore, tandem repeats of 3 WH2 (V, or more recently also W) domains render JMY capable of Arp2/3 independent actin assembly. The motility promoting cytoplasmic function of JMY is abrogated upon DNA-damage and nuclear translocation of JMY. To address the precise cellular function of JMY in cellular actin rearrangements, we have searched for potential new interaction partners by mass spectrometry. We identified several candidates and correlated their localization with the subcellular dynamics of JMY. JMY is localized to dynamic vesiculo-tubular structures throughout the cytoplasm, which are decorated with actin and Arp2/3 complex. Moreover, JMY partially colocalizes and interacts with VAP-A, which is involved in vesicle-based transport processes. Finally, overexpression of JMY results in Golgi dispersal by loss from the trans-site and affects VSV-G transport. These analyses, together with biochemical experiments, indicate that JMY drives vesicular trafficking in the trans-Golgi region and at ER-membrane contact sites (MCS), distinct from other Arp2/3 activators involved in vesicle transport processes such as the related WHAMM or WASH.

Arp2/3 complex is essential for actin network treadmilling as well as for targeting of capping protein and cofilin.

Lamellipodia are sheet-like protrusions formed during migration or phagocytosis and comprise a network of actin filaments. Filament formation in this network is initiated by nucleation/branching through the actin-related protein 2/3 (Arp2/3) complex downstream of its activator, suppressor of cAMP receptor/WASP-family verprolin homologous (Scar/WAVE), but the relative relevance of Arp2/3-mediated branching versus actin filament elongation is unknown. Here we use instantaneous interference with Arp2/3 complex function in live fibroblasts with established lamellipodia. This allows direct examination of both the fate of elongating filaments upon instantaneous suppression of Arp2/3 complex activity and the consequences of this treatment on the dynamics of other lamellipodial regulators. We show that Arp2/3 complex is an essential organizer of treadmilling actin filament arrays but has little effect on the net rate of actin filament turnover at the cell periphery. In addition, Arp2/3 complex serves as key upstream factor for the recruitment of modulators of lamellipodia formation such as capping protein or cofilin. Arp2/3 complex is thus decisive for filament organization and geometry within the network not only by generating branches and novel filament ends, but also by directing capping or severing activities to the lamellipodium. Arp2/3 complex is also crucial to lamellipodia-based migration of keratocytes.

Structural basis for complex formation between human IRSp53 and the translocated intimin receptor Tir of enterohemorrhagic E. coli.

Actin assembly beneath enterohemorrhagic E. coli (EHEC) attached to its host cell is triggered by the intracellular interaction of its translocated effector proteins Tir and EspF(U) with human IRSp53 family proteins and N-WASP. Here, we report the structure of the N-terminal I-BAR domain of IRSp53 in complex with a Tir-derived peptide, in which the homodimeric I-BAR domain binds two Tir molecules aligned in parallel. This arrangement provides a protein scaffold linking the bacterium to the host cell's actin polymerization machinery. The structure uncovers a specific peptide-binding site on the I-BAR surface, conserved between IRSp53 and IRTKS. The Tir Asn-Pro-Tyr (NPY) motif, essential for pedestal formation, is specifically recognized by this binding site. The site was confirmed by mutagenesis and in vivo-binding assays. It is possible that IRSp53 utilizes the NPY-binding site for additional interactions with as yet unknown partners within the host cell.

Microtubules as platforms for assaying actin polymerization in vivo.

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.