How kinetochore proteins form a dynamic interface with microtubules is largely unknown. In budding yeast, the 10-protein Dam1 complex is an Aurora kinase target that plays essential roles maintaining the integrity of the mitotic spindle and regulating interactions with the kinetochore. Here, we investigated the biochemical properties of purified Dam1 complex. The complex oligomerized into rings around microtubules. Ring formation was facilitated by microtubules but could occur in their absence. Mutant alleles led to partially assembled complexes or reduced microtubule binding. The interaction between rings and microtubules is mediated by the C termini of both Dam1 and alphabeta-tubulin. Ring formation promotes microtubule assembly, stabilizes against disassembly, and promotes bundling. A GTP-tubulin lattice is the preferred binding partner for the complex, and Dam1 rings can exhibit lateral mobility on microtubules. These observations suggest a mechanism by which the kinetochore can recognize and stay attached to the plus ends of microtubules.

The Arp2/3 complex is a seven-protein assembly that is critical for actin nucleation and branching in cells. Here we report the reconstitution of active human Arp2/3 complex after expression of all seven subunits in insect cells. Expression of partial complexes revealed that a heterodimer of the p34 and p20 subunits constitutes a critical structural core of the complex, whereas the remaining subunits are peripherally located. Arp3 is crucial for nucleation, consistent with it being a structural component of the nucleation site. p41, p21, and p16 contribute differently to nucleation and stimulation by ActA and WASP, whereas p34/p20 bind actin filaments and likely function in actin branching. This study reveals that the nucleating and organizing functions of Arp2/3 complex subunits are separable, indicating that these activities may be differentially regulated in cells.

Molecular mechanisms for cell migration, especially how signaling and cytoskeletal systems are integrated, are not understood well. Here, we examined the role of CARMIL (capping protein, Arp2/3, and Myosin-I linker) family proteins in migrating cells. Vertebrates express three conserved genes for CARMIL, and we examined the functions of the two CARMIL genes expressed in migrating human cultured cells. Both isoforms, CARMIL1 and 2, were necessary for cell migration, but for different reasons. CARMIL1 localized to lamellipodia and macropinosomes, and loss of its function caused loss of lamellipodial actin, along with defects in protrusion, ruffling, and macropinocytosis. CARMIL1-knockdown cells showed loss of activation of Rac1, and CARMIL1 was biochemically associated with the GEF Trio. CARMIL2, in contrast, colocalized with vimentin intermediate filaments, and loss of its function caused a distinctive multipolar phenotype. Loss of CARMIL2 also caused decreased levels of myosin-IIB, which may contribute to the polarity phenotype. Expression of one CARMIL isoform was not able to rescue the knockdown phenotypes of the other. Thus, the two isoforms are both important for cell migration, but they have distinct functions.

CD2-associated protein (CD2AP) is a scaffold molecule that plays a critical role in the maintenance of the kidney filtration barrier. Little, however, is understood about its mechanism of function. We used mass spectrometry to identify CD2AP-interacting proteins. Many of the proteins that we identified suggest a role for CD2AP in endocytosis and actin regulation. To address the role of CD2AP in regulation of the actin cytoskeleton, we focused on characterizing the interaction of CD2AP with actin-capping protein CP. We identified a novel binding motif LXHXTXXRPK(X) 6 P present in CD2AP that is also found in its homolog Cin85 and other capping protein-associated proteins such as CARMIL and CKIP-1. CD2AP inhibits the function of capping protein in vitro. Therefore, our results support a role of CD2AP in the regulation of the actin cytoskeleton. CD2-associated protein (CD2AP)2 is a 70-kDa protein that was originally cloned as a protein that interacts with the cytoplasmic tail of CD2, a T lymphocyte and natural killer cell transmembrane protein (1). It is composed of three Src homology 3 (SH3) domains at the NH 2 terminus followed by proline-rich sequences and a coiled-coil domain at the extreme COOH terminus. It is expressed in all tissues except brain. Interestingly, CD2AP-deficient animals die of renal failure ϳ6 weeks of age (2). In the kidney, CD2AP is highly expressed in the glomerular epithelial cell, and it is implicated to play a role in a specialized cell junction known as a slit diaphragm (3).A homolog of CD2AP, Cin85, was cloned as an interacting protein with the E3 ubiquitin ligase c-cbl (4) and as an inhibitor of phosphatidylinositol 3-kinase (5). Recently, several endocytic and actin-associated molecules have been reported to interact with CD2AP and Cin85. Some proteins have been demonstrated to interact with both CD2AP and Cin85, whereas others have only been shown to bind one or the other. CD2AP has been shown to play a role in vesicular trafficking because of interactions with c-cbl and an active form of Rab4 (6). Cin85 has also been shown to bind to molecules involved in endocytosis, such as endophilin, synaptojanin 2B1, and SHIP-1 and the clathrin scaffold HIP1R (7,8). Both CD2AP and Cin85 contain a motif, FXDXF, that mediates interactions with the ␣-appendage of clathrin adaptor protein 2 (9). Interactions of CD2AP and Cin85 with the phosphatidylinositol bisphosphate-dependent GTPase for ARF1 and ARF5, known as ASAP1, as well as cortactin-and actin-capping protein suggest additional roles in the regulation of the actin cytoskeleton (7, 10 -12).To further elucidate the molecular mechanism of CD2AP function, we performed mass spectrometry to identify interacting proteins. We identified novel and previously known interacting proteins such as actin-capping protein CP (11).Over the last decade, there has been much progress in our understanding of how the actin cytoskeleton is regulated. Critical is the polymerization of monomeric G-actin to forming an asymmetric actin filament with a barbed and a poin...

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.