R. Dyche Mullins

University of California, San Francisco | UCSF · Department of Cellular and Molecular Pharmacology

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes, including phagocytosis, endocytosis, and pseudopod protrusion. When confronted with opposing forces, the growth rate of these networks slows and their density increases, but the stoichiometry of key components doe...

Significant technical challenges have limited the study of extremophile cell biology. Here we describe a system for imaging samples at 75°C using high numerical aperture, oil-immersion lenses. With this system we observed and quantified the dynamics of cell division in the model thermoacidophilic crenarchaeon Sulfolobus acidocaldarius with unpreced...

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes. Load forces slow the growth and increase the density of these networks, but the molecular mechanisms governing this force response are not well understood. Here we use single-molecule imaging and AFM cantilever de...

The shape of many eukaryotic cells depends on the actin cytoskeleton, and changes in actin assembly dynamics underlie many changes in cell shape. Ena/VASP-family actin polymerases, for example, modulate cell shape by locally accelerating actin filament assembly and slowing filament capping. When concentrated into discrete foci at the leading edge,...

P values and error bars help readers infer whether a reported difference would likely recur, with the sample size n used for statistical tests representing biological replicates, independent measurements of the population from separate experiments. We provide examples and practical tutorials for creating figures that communicate both the cell-level...

Cell biology is littered with erroneously tiny P values, often the result of evaluating individual cells as independent samples. Because readers expect low P values and small error bars to imply that an observed difference would persist if the experiment were to be duplicated, the sample size (N) used for statistical tests should actually be the nu...

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates....

During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic...

The actin cytoskeleton comprises a set of filament networks that perform essential functions in eukaryotic cells. The idea that actin filaments incorporate monomers directly from solution forms both the “textbook picture” of filament elongation and a conventional starting point for quantitative modeling of cellular actin dynamics. Recent work, howe...

During autophagy actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in non-starved cells, cytoplasmic...

The actin cytoskeleton comprises a set of filament networks that perform essential functions in eukaryotic cells. The idea that actin filaments incorporate monomers directly from solution forms both the “textbook picture” of filament elongation and a conventional starting point for quantitative modeling of cellular actin dynamics. Recent work, howe...

The actin cytoskeleton comprises a set of filament networks that perform essential functions in eukaryotic cells. The idea that actin filaments incorporate monomers directly from solution forms both the “textbook picture” of filament elongation and a conventional starting point for quantitative modeling of cellular actin dynamics. Recent work, howe...

Significance Actin filaments are dynamic cytoskeletal elements that assemble upon ATP binding. Actin homologs are present in all domains of life, and all share a similar 3D structure of the assembling subunit, but evolutionary changes to the subunit have generated many different actin filament structures. The filament structure of the bacterial act...

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WASP-family proteins are known to promote assembly of branched actin networks by stimulating the filament-nucleating activity of the Arp2/3 complex. Here, we show that WASP-family proteins also function as polymerases that accelerate elongation of uncapped actin filaments. When clustered on a surface, WASP-family proteins can drive branched actin n...

‘cell protrusions analysis functions.R’ Analysis script for use in R to calculate and plot the path characteristics, and the relationships with pseudopod activity.

‘protrusions_chimera_script.py’ Python script used to calculate protrusion volumes with UCSF chimera. This outputs two text files: one containing data for pseudopod volumes at each time point and one of the angles between the main pseudopod and the cell path.

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partiti...

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods...

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α- and β-tubulin, called BtubA and BtubB (BtubA/B), that have been observed to assemble into filaments in the presence of GTP. BtubA/B polymers are proposed to be composed in vitro by two to six protofilaments in contrast to that in vivo, where they have been reported to form 5-pr...

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode and identify its phenotypic and molecular markers. In this study, we focus on a widely dispersed migration mode characterized by dynamic actin-filled ps...

Diverse eukaryotic cells crawl through complex environments using distinct modes of migration. To understand the underlying mechanisms and their evolutionary relationships, we must define each mode, and identify its phenotypic and molecular markers. Here, we focus on a widely dispersed migration mode characterized by dynamic, actin-filled pseudopod...

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods...

Bacteria of the genus Prosthecobacter express homologs of eukaryotic α-and β-tubulin, called BtubA and BtubB, that have been observed to assemble into bacterial microtubules (bMTs). The btubAB genes likely entered the Prosthecobacter lineage via horizontal gene transfer and may derive from an early ancestor of the modern eukaryotic microtubule (MT)...

Dynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copy-number plasmid pLS20 in Bacillus subtilis. Purified Alp7A forms dynamically unstab...

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composi...

Branched actin networks—created by the Arp2/3 complex, capping protein, and a nucleation promoting factor—generate and transmit forces required for many cellular processes, but their response to force is poorly understood. To address this, we assembled branched actin networks in vitro from purified components and used simultaneous fluorescence and...

Fluorescent derivatives of actin and actin-binding domains are powerful tools for studying actin filament architecture and dynamics in live cells. Growing evidence, however, indicates that these probes are biased, and their cellular distribution does not accurately reflect that of the cytoskeleton. To understand the strengths and weaknesses of comm...

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowt...

Custom ImageJ macros. DOI: http://dx.doi.org/10.7554/eLife.07735.019

Tropomyosins are coiled-coil proteins that bind actin filaments and regulate multiple cytoskeletal functions, including actin network dynamics near the leading edge of motile cells. Previous work demonstrated that tropomyosins inhibit actin nucleation by the Arp2/3 complex and prevent filament disassembly by cofilin. We find that the Arp2/3 complex...

Most eukaryotic cells express multiple isoforms of the actin-binding protein tropomyosin that help construct a variety of cytoskeletal networks. Only one non-muscle tropomyosin (Tm1A) has previously been described in Drosophila, but developmental defects caused by insertion of P-elements near tropomyosin genes imply the existence of additional, non...

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hu...

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroar...

The sequence, structure, and assembly dynamics of eukaryotic actins are conserved across phyla. In contrast, actin-like proteins (ALPs) from eubacteria share little sequence homology, form polymers with different architectures, and assemble with different kinetics. The structural and functional diversity of the bacterial ALPs appears to arise from...

Interactions between antiparallel microtubules are essential for the organization of spindles in dividing cells. The ability to form immobilized antiparallel microtubule pairs in vitro, combined with the ability to image them via TIRF microscopy, permits detailed biochemical characterization of microtubule cross-linking proteins and their effects o...

Significance Many bacteria contain large, circular DNA molecules, called plasmids, that encode physiologically, medically, and commercially important genes, including genes conferring virulence and drug resistance. The largest plasmids use active segregation systems to maintain themselves in the host bacterium. Such segregation systems provide rema...

A major aim of our work is to understand the mechanisms behind dynamic organization of the cellular plasma membrane, especially local heterogeneities such as nanometer sized lipid domains (Mayor and Rao, 2004). As reported previously, glycosyl-phosphatidylinositol-anchored protein (GPI-AP) organization in nano-clusters in the plasma membrane is dri...

Branched actin networks generate protrusive forces required for cell motility and movement of sub-cellular structures. While biochemical knowledge of dendritic actin network assembly continues to advance, relatively little is known about how the mechanical properties of networks respond to physical stimuli during growth. By combining surface microp...

The actin-binding protein αE-catenin may contribute to transitions between cell migration and cell-cell adhesion that depend on remodeling the actin cytoskeleton, but the underlying mechanisms are unknown. We show that the αE-catenin actin-binding domain (ABD) binds cooperatively to individual actin filaments, and that binding is accompanied by a c...

In the cytoplasm, actin filaments form crosslinked networks that enable eukaryotic cells to transport cargo, change shape, and move. Actin is also present in the nucleus but, in this compartment, its functions are more cryptic and controversial. If we distill the substantial literature on nuclear actin down to its essentials, we find four, recurrin...

The actin cytoskeleton is essential to all eukaryotic cells. In addition to playing important structural roles, assembly of actin into filaments powers diverse cellular processes, including cell motility, cytokinesis, and endocytosis. Actin polymerization is tightly regulated by its numerous cofactors, which control spatial and temporal assembly of...

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits e...

In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or non-canonical oligomer) of nuclear actin are hotly debated and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and to e...

Actin-related proteins (Arps) are a class of proteins found in all eukaryotes and many species of bacteria and archaea. Arps are defined by their degree of similarity to actin (conventional actin), a ubiquitous, eukaryotic cytoskeletal protein. All Arps are built around a common structural fold. This actin fold binds and hydrolyzes adenosine tripho...

Modern research and training in the life sciences require cross-disciplinary programs, integrating concepts and methods from biology, physics, chemistry, and mathematics. We describe the structure and outcomes from an example of one such approach, the Physiology Course at the Marine Biological Laboratory (MBL) in Woods Hole, Massachusetts, and disc...

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion...

Actin filaments and microtubules control the shape of cells and organize their contents. The proteins that form these cytoskeletal polymers were identified and isolated decades ago—actin in 1942 (1) and tubulin in 1968 (2)—but we still argue about some of their basic properties. New work from Niedermayer et al. (3), however, puts us on the right tr...

Junction-mediating and regulatory protein (JMY) is a regulator of both transcription and actin filament assembly. In response to DNA damage, JMY accumulates in the nucleus and promotes p53-dependent apoptosis. JMY's actin-regulatory activity relies on a cluster of three actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domains that nu...

To establish and maintain their internal organization, living cells must move molecules to their correct locations. Long-range intracellular movements are often driven by motor molecules moving along microtubules, similarly to trucks driving along a highway. Recent work demonstrates that some randomly dispersed cargos can generate actin filaments t...