INF2 mutations cause kidney disease through a gain-of-function mechanism.

Heterozygosity for inverted formin-2 (INF2) mutations causes focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth disease. A key question is whether the disease is caused by gain-of-function effects on INF2 or loss of function (haploinsufficiency). Despite established roles in multiple cellular processes, neither INF2 knockout mice nor mice with a disease-associated point mutation display an evident kidney or neurologic phenotype.

Missense Mutant Gain-of-Function Causes Inverted Formin 2 (INF2)-Related Focal Segmental Glomerulosclerosis (FSGS).

Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice.

Fatty acyl-coenzyme A activates mitochondrial division through oligomerization of MiD49 and MiD51.

Mitochondrial fission occurs in many cellular processes, but the regulation of fission is poorly understood. We show that long-chain acyl-coenzyme A (LCACA) activates two related mitochondrial fission proteins, MiD49 and MiD51, by inducing their oligomerization, which activates their ability to stimulate the DRP1 GTPase. The 1:1 stoichiometry of LCACA:MiD in the oligomer suggests interaction in the previously identified nucleotide-binding pocket, and a point mutation in this pocket reduces LCACA binding and LCACA-induced oligomerization for MiD51.

Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin.

Drp1 is a dynamin family GTPase required for mitochondrial and peroxisomal division. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600.

Effects of phosphorylation on Drp1 activation by its receptors, actin, and cardiolipin.

Drp1 is a dynamin family GTPase that is required for mitochondrial and peroxisomal division, in which it oligomerizes into a ring and constricts the underlying membrane in a GTP hydrolysis-dependent manner. Oligomerization increases Drp1 GTPase activity through interactions between neighboring GTPase domains. In cells, Drp1 is regulated by several factors including Drp1 receptors, actin filaments, cardiolipin, and phosphorylation at two sites: S579 and S600.

The multiple links between actin and mitochondria.

Actin plays many well-known roles in cells, and understanding any specific role is often confounded by the overlap of multiple actin-based structures in space and time. Here, we review our rapidly expanding understanding of actin in mitochondrial biology, where actin plays multiple distinct roles, exemplifying the versatility of actin and its functions in cell biology. One well-studied role of actin in mitochondrial biology is its role in mitochondrial fission, where actin polymerization from the endoplasmic reticulum through the formin INF2 has been shown to stimulate two distinct steps.

Acute ACAT1/SOAT1 Blockade Increases MAM Cholesterol and Strengthens ER-Mitochondria Connectivity.

Cholesterol is a key component of all mammalian cell membranes. Disruptions in cholesterol metabolism have been observed in the context of various diseases, including neurodegenerative disorders such as Alzheimer's disease (AD). The genetic and pharmacological blockade of acyl-CoA:cholesterol acyltransferase 1/sterol O-acyltransferase 1 (ACAT1/SOAT1), a cholesterol storage enzyme found on the endoplasmic reticulum (ER) and enriched at the mitochondria-associated ER membrane (MAM), has been shown to reduce amyloid pathology and rescue cognitive deficits in mouse models of AD.

INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury.

During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines.

Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation.

Mitochondrial damage represents a dramatic change in cellular homeostasis. One rapid response is perimitochondrial actin polymerization, termed acute damage-induced actin (ADA). The consequences of ADA are not understood.

Myosin II proteins are required for organization of calcium-induced actin networks upstream of mitochondrial division.

The formin INF2 polymerizes a calcium-activated cytoplasmic network of actin filaments, which we refer to as calcium-induced actin polymerization (CIA). CIA plays important roles in multiple cellular processes, including mitochondrial dynamics and vesicle transport. Here, we show that nonmuscle myosin II (NMII) is activated within 60 s of calcium stimulation and rapidly recruited to the CIA network.

Parallel kinase pathways stimulate actin polymerization at depolarized mitochondria.

Mitochondrial damage (MtD) represents a dramatic change in cellular homeostasis, necessitating metabolic changes and stimulating mitophagy. One rapid response to MtD is a rapid peri-mitochondrial actin polymerization termed ADA (acute damage-induced actin). The activation mechanism for ADA is unknown.

Mff oligomerization is required for Drp1 activation and synergy with actin filaments during mitochondrial division.

Mitochondrial division is an important cellular process in both normal and pathological conditions. The dynamin GTPase Drp1 is a central mitochondrial division protein, driving constriction of the outer mitochondrial membrane (OMM). In mammals, the OMM protein mitochondrial fission factor (Mff) is a key receptor for recruiting Drp1 from the cytosol to the mitochondrion.

Multiple roles for actin in secretory and endocytic pathways.

Actin filaments play multiple roles in the secretory pathway and in endosome dynamics in mammals, including maintenance of Golgi structure, release of membrane cargo from the trans-Golgi network (TGN), endocytosis, and endosomal sorting dynamics. In addition, TGN carrier transport and endocytosis both occur by multiple mechanisms in mammals. Actin likely plays a role in at least four mammalian endocytic pathways, five pathways for membrane release from the TGN, and three processes involving endosomes.

Revolutionary view of two ways to split a mitochondrion.

Organelles called mitochondria divide in at least two contexts: during cell growth and in response to mitochondrial damage. The finding that division is different in these two contexts sheds light on the regulatory pathways involved.

SEC24A facilitates colocalization and Ca flux between the endoplasmic reticulum and mitochondria.

A genome-wide screen recently identified SEC24A as a novel mediator of thapsigargin-induced cell death in HAP1 cells. Here, we determined the cellular mechanism and specificity of SEC24A-mediated cytotoxicity. Measurement of Ca levels using organelle-specific fluorescent indicator dyes showed that Ca efflux from endoplasmic reticulum (ER) and influx into mitochondria were significantly impaired in -knockout cells.

Lysine acetylation of cytoskeletal proteins: Emergence of an actin code.

Reversible lysine acetylation of nuclear proteins such as histones is a long-established important regulatory mechanism for chromatin remodeling and transcription. In the cytoplasm, acetylation of a number of cytoskeletal proteins, including tubulin, cortactin, and the formin mDia2, regulates both cytoskeletal assembly and stability. More recently, acetylation of actin itself was revealed to regulate cytoplasmic actin polymerization through the formin INF2, with downstream effects on ER-to-mitochondrial calcium transfer, mitochondrial fission, and vesicle transport.

Tumor microtubes connect pancreatic cancer cells in an Arp2/3 complex-dependent manner.

Actin-based tubular connections between cells have been observed in many cell types. Termed "tunneling nanotubes (TNTs)," "membrane nanotubes," "tumor microtubes (TMTs)," or "cytonemes," these protrusions interconnect cells in dynamic networks. Structural features in these protrusions vary between cellular systems, including tubule diameter and the presence of microtubules.

FSGS-Causing INF2 Mutation Impairs Cleaved INF2 N-Fragment Functions in Podocytes.

Background: Mutations in the gene encoding inverted formin-2 (INF2), a member of the formin family of actin regulatory proteins, are among the most common causes of autosomal dominant FSGS. INF2 is regulated by interaction between its N-terminal diaphanous inhibitory domain (DID) and its C-terminal diaphanous autoregulatory domain (DAD). INF2 also modulates activity of other formins, such as the mDIA subfamily, and promotes stable microtubule assembly.

Regulation of INF2-mediated actin polymerization through site-specific lysine acetylation of actin itself.

INF2 is a formin protein that accelerates actin polymerization. A common mechanism for formin regulation is autoinhibition, through interaction between the N-terminal diaphanous inhibitory domain (DID) and C-terminal diaphanous autoregulatory domain (DAD). We recently showed that INF2 uses a variant of this mechanism that we term "facilitated autoinhibition," whereby a complex consisting of cyclase-associated protein (CAP) bound to lysine-acetylated actin (KAc-actin) is required for INF2 inhibition, in a manner requiring INF2-DID.

Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors.

Recent studies show that mitochondria and actin filaments work together in two contexts: (1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission and (2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min).

A complex containing lysine-acetylated actin inhibits the formin INF2.

Inverted formin 2 (INF2) is a member of the formin family of actin assembly factors. Dominant missense mutations in INF2 are linked to two diseases: focal segmental glomerulosclerosis, a kidney disease, and Charcot-Marie-Tooth disease, a neuropathy. All of the disease mutations map to the autoinhibitory diaphanous inhibitory domain.

The Pollard lab at Salk: moving the leading edge forward.

This essay will review the years that the Pollard lab was at the Salk Institute in the last half of the 1990s. It was a highly productive time both in research and in training. For me personally, it shaped my career for the better in ways I am still discovering.

Roles for Ena/VASP proteins in FMNL3-mediated filopodial assembly.

Filopodia are actin-dependent finger-like structures that protrude from the plasma membrane. Actin filament barbed-end-binding proteins localized to filopodial tips are key to filopodial assembly. Two classes of barbed-end-binding proteins are formins and Ena/VASP proteins, and both classes have been localized to filopodial tips in specific cellular contexts.

Long-Term Potentiation Requires a Rapid Burst of Dendritic Mitochondrial Fission during Induction.

Synaptic transmission is bioenergetically demanding, and the diverse processes underlying synaptic plasticity elevate these demands. Therefore, mitochondrial functions, including ATP synthesis and Ca handling, are likely essential for plasticity. Although axonal mitochondria have been extensively analyzed, LTP is predominantly induced postsynaptically, where mitochondria are understudied.