The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion

Abalance between fission and fusion events determines the morphology of mitochondria. In yeast, mitochondrial fission is regulated by the outer membrane–associated dynamin-related GTPase, Dnm1p. Mitochondrial fusion requires two integral outer membrane components, Fzo1p and Ugo1p. Interestingly, mutations in a second mitochondrial-associated dynamin-related GTPase, Mgm1p, produce similar phenotypes to fzo1 and ugo cells. Specifically, mutations in MGM1 cause mitochondrial fragmentation and a loss of mitochondrial DNA that are suppressed by abolishing DNM1-dependent fission. In contrast to fzo1ts mutants, blocking DNM1-dependent fission restores mitochondrial fusion in mgm1ts cells during mating. Here we show that blocking DNM1-dependent fission in Δmgm1 cells fails to restore mitochondrial fusion during mating. To examine the role of Mgm1p in mitochondrial fusion, we looked for molecular interactions with known fusion components. Immunoprecipitation experiments revealed that Mgm1p is associated with both Ugo1p and Fzo1p in mitochondria, and that Ugo1p and Fzo1p also are associated with each other. In addition, genetic analysis of specific mgm1 alleles indicates that Mgm1p's GTPase and GTPase effector domains are required for its ability to promote mitochondrial fusion and that Mgm1p self-interacts, suggesting that it functions in fusion as a self-assembling GTPase. Mgm1p's localization within mitochondria has been controversial. Using protease protection and immuno-EM, we have shown previously that Mgm1p localizes to the intermembrane space, associated with the inner membrane. To further test our conclusions, we have used a novel method using the tobacco etch virus protease and confirm that Mgm1p is present in the intermembrane space compartment in vivo. Taken together, these data suggest a model where Mgm1p functions in fusion to remodel the inner membrane and to connect the inner membrane to the outer membrane via its interactions with Ugo1p and Fzo1p, thereby helping to coordinate the behavior of the four mitochondrial membranes during fusion

The elementary events underlying force generation in neuronal lamellipodia

We have used optical tweezers to identify the elementary events underlying force generation in neuronal lamellipodia. When an optically trapped bead seals on the lamellipodium membrane, Brownian fluctuations decrease revealing the underlying elementary events. The distribution of bead velocities has long tails with frequent large positive and negative values associated to forward and backward jumps occurring in 0.1–0.2 ms with varying amplitudes up to 20 nm. Jump frequency and amplitude are reduced when actin turnover is slowed down by the addition of 25 nM Jasplakinolide. When myosin II is inhibited by the addition of 20 μM Blebbistatin, jump frequency is reduced but to a lesser extent than by Jasplainolide. These jumps constitute the elementary events underlying force generation

Expression of Actin-interacting Protein 1 Suppresses Impaired Chemotaxis of Dictyostelium Cells Lacking the Na+-H+ Exchanger NHE1

Dictyostelium cells lacking the intracellular pH regulator NHE1 have defective chemotaxis. A modifier screen and reconstitution studies show expression of recombinant actin interacting protein 1 (Aip1) suppresses the Ddnhe1-phenotype. Aip1 promotes cofilin-dependent actin remodeling, which is likely a major determinant in pH-dependent chemotaxis

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

Bayesian Modeling of the Yeast SH3 Domain Interactome Predicts Spatiotemporal Dynamics of Endocytosis Proteins

A genome-scale specificity and interaction map for yeast SH3 domain-containing proteins reveal how family members show selective binding to target proteins and predicts the dynamic localization of new candidate endocytosis proteins

The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins

The accuracy of tail-anchored (TA) protein targeting to the endoplasmic reticulum (ER) depends on the Guided Entry of Tail-Anchored (Get) protein targeting machinery. The fate of TA proteins that become inappropriately inserted into other organelles, such as mitochondria, is unknown. Here, we identify Msp1, a conserved, membrane-anchored AAA-ATPase (ATPase associated with a variety of cellular activities) that localizes to mitochondria and peroxisomes, as a critical factor in a quality control pathway that senses and degrades TA proteins mistargeted to the outer mitochondrial membrane (OMM). Pex15 is normally targeted by the Get pathway to the ER, from where it travels to peroxisomes. Loss of Msp1 or loss of the Get pathway results in the redistribution of Pex15 to mitochondria. Cells lacking both a functional Get pathway and Msp1 accumulate increased amounts of Pex15 on the OMM and display severely dysfunctional mitochondrial morphology. In addition, Msp1 binds and promotes the turnover of a Pex15 mutant that is misdirected to the OMM. Our data suggest that Msp1 functions in local organelle surveillance by extracting mistargeted proteins, ensuring the fidelity of organelle specific-localization of TA proteins

Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We report time courses of the accumulation and loss of 16 fluorescent fusion proteins at sites of clathrin-mediated endocytosis in fission yeast. Mathematical modeling shows that dendritic nucleation hypothesis can account for the kinetics of actin assembly in vivo and disassembly requires actin filament severing along with depolymerization

Turnover of branched actin filament networks by stochastic fragmentation with ADF/cofilin

During actin-based motility, capping protein influences actin-depolymerizing factor (ADF)/cofilin binding that increases in space and time concomitant with the state of the nucleotide associated to the actin subunits. ADF/cofilin induces microscopic fragmentation of the actin network, which induces turnover through a macroscopic, stochastic dynamic mechanism