Biology subjects

Pimm, M. L.

Publications and source records attributed to Pimm, M. L..

6 recordsLinked to original sources

bioRxiv2026

Constitutively active mDia1 enables visualization of actin assembly in filopodia

Visualizing individual actin filaments in living cells is notoriously difficult. Formins, which track filament plus ends, are powerful probes of actin assembly, yet their behavior in cells is incompletely understood. We dissect the formin mDia1 to reveal how regulatory states control filament assembly. Using TIRF microscopy, we directly compare new regulatory mutants with canonical activity constructs (full-length, FH1-C, FH2-C, and {Delta}DAD). A two amino-acid substitution mutant, mDia1(CA), is constitutively active and drives strong actin nucleation and elongation. In cells, SNAP-mDia1(CA) is enriched in filopodia, where it forms bright, persistent signals at the tips and dim puncta in the shafts. SNAP-mDia1(CA) increases filopodia number but not elongation rate or length, regardless of its use as an overexpression or rescue construct. Although mDia1(CA) is not a universal actin plus-end tracker in this system, its selective enrichment at filopodial plus ends establishes it as a mechanistically distinct probe for interrogating filopodial actin dynamics.

cell biology↗

bioRxiv2025

Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

Microtubules are cytoskeletal filaments that self-assemble from the protein tubulin, a heterodimer of -tubulin and {beta}-tubulin, and are important for cell mechanics, migration, and division. Much work has focused on how the nucleotide state of {beta}-tubulin regulates the structure and dynamics of microtubules. In contrast, less is known about the structure and function of the C-terminal tails (CTTs) of - and {beta}-tubulin which are thought to freely protrude from the surface of the microtubule. To study the CTT of -tubulin, we developed three different biosensors that bind the tyrosinated -tubulin CTT (Y-CTT). Surprisingly, live imaging of the probes indicates that the Y-CTT is minimally accessible along the microtubule lattice under normal cellular conditions. Lattice binding of the Y-CTT probes can be increased by three different ways of changing the tubulin conformational state: the drug Taxol, expression of microtubule-associated proteins (MAPs) that recognize or promote an expanded tubulin conformation, or expression of tubulin that cannot hydrolyze GTP. Molecular dynamics simulations indicate that the Y-CTT undergoes numerous transient interactions with the bodies of -tubulin and {beta}-tubulin in the lattice, and that the frequency of these interactions is regulated by the tubulin nucleotide state. These findings suggest that accessibility of the Y-CTT is locally governed by nucleotide- and MAP-dependent conformational changes to tubulin subunits within the microtubule lattice.

cell biology↗

bioRxiv2023

Coordination of actin plus-end dynamics by IQGAP1, formin, and capping protein

Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (Capping Protein, CPz). We explore IQGAP1s roles regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF we show that IQ-GAP1s displacement activity extends to formin-CPz decision complexes, promoting end-binding protein turnover at plusends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.

cell biology↗

bioRxiv2022

Purification of Human β and γ Actin from Budding Yeast

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified -actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins and there is an underlying concern that these proteins perform differently with actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human {beta}- or {gamma}-actin (i.e., cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both {beta}- or {gamma}-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin, and thymosin-{beta}4 (T{beta}4). Notably, T{beta}4 and profilin bind to {beta}- or {gamma}-actin with higher affinity than to -actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies of actin regulation.

biochemistry↗

bioRxiv2022

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin-mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-PLP interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.

biochemistry↗

bioRxiv2021

Functional fluorescently-tagged human profilin

Profilin-1 (PFN1) is a cytoskeletal protein that regulates the dynamics of actin and microtubule assembly. Thus, PFN1 is essential for the normal division, motility, and morphology of cells. Unfortunately, conventional fusion and direct labeling strategies compromise different facets of PFN1 function. As a consequence, the only methods used to determine known PFN1 functions have been indirect and often deduced in cell-free biochemical assays. We engineered and characterized two genetically encoded versions of tagged PFN1 that behave identical to each other and the tag-free protein. In biochemical assays purified proteins bind to PIP lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and impact microtubule dynamics. Halo-PFN1 or mApple-PFN1 restored morphological and cytoskeletal functions in PFN1-deficient mammalian cells. In biochemical assays, mAp-PFN1 bound tubulin dimers (kD = 1.89 M) and the sides of microtubules in vitro. Titrations of self-labeling Haloligands were used to visualize molecules of PFN1. This approach combined with specific function-disrupting point-mutants (Y6D and R88E) revealed PFN1 bound to microtubules in live cells. Cells expressing the ALS-associated G118V disease variant did not associate with actin filaments or microtubules. Thus, these tagged PFN1s are reliable tools for studying the dynamic interactions of PFN1 with actin or microtubules in vitro as well as in important cell processes or disease-states.

cell biology↗