Biology subjects

Haarer, B. K.

Publications and source records attributed to Haarer, B. K..

3 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↗

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↗