Purification of human ß- and γ-actin from budding yeast.

Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified α-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human ß- or γ-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both ß- or γ-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 (formin), fascin and thymosin-ß4 (Tß4). Notably, Tß4 and profilin bind to ß- or γ-actin with higher affinity than to α-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 on actin regulation.

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis.

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion.

ACTIN DEPOLYMERIZING FACTOR4 regulates actin dynamics during innate immune signaling in Arabidopsis.

Conserved microbe-associated molecular patterns (MAMPs) are sensed by pattern recognition receptors (PRRs) on cells of plants and animals. MAMP perception typically triggers rearrangements to actin cytoskeletal arrays during innate immune signaling. However, the signaling cascades linking PRR activation by MAMPs to cytoskeleton remodeling are not well characterized. Here, we developed a system to dissect, at high spatial and temporal resolution, the regulation of actin dynamics during innate immune signaling in plant cells. Within minutes of MAMP perception, we detected changes to single actin filament turnover in epidermal cells treated with bacterial and fungal MAMPs. These MAMP-induced alterations phenocopied an ACTIN DEPOLYMERIZING FACTOR4 (ADF4) knockout mutant. Moreover, actin arrays in the adf4 mutant were unresponsive to a bacterial MAMP, elf26, but responded normally to the fungal MAMP, chitin. Together, our data provide strong genetic and cytological evidence for the inhibition of ADF activity regulating actin remodeling during innate immune signaling. This work is the first to directly link an ADF/cofilin to the cytoskeletal rearrangements elicited directly after pathogen perception in plant or mammalian cells.

The plant actin cytoskeleton responds to signals from microbe-associated molecular patterns.

Plants are constantly exposed to a large and diverse array of microbes; however, most plants are immune to the majority of potential invaders and susceptible to only a small subset of pathogens. The cytoskeleton comprises a dynamic intracellular framework that responds rapidly to biotic stresses and supports numerous fundamental cellular processes including vesicle trafficking, endocytosis and the spatial distribution of organelles and protein complexes. For years, the actin cytoskeleton has been assumed to play a role in plant innate immunity against fungi and oomycetes, based largely on static images and pharmacological studies. To date, however, there is little evidence that the host-cell actin cytoskeleton participates in responses to phytopathogenic bacteria. Here, we quantified the spatiotemporal changes in host-cell cytoskeletal architecture during the immune response to pathogenic and non-pathogenic strains of Pseudomonas syringae pv. tomato DC3000. Two distinct changes to host cytoskeletal arrays were observed that correspond to distinct phases of plant-bacterial interactions i.e. the perception of microbe-associated molecular patterns (MAMPs) during pattern-triggered immunity (PTI) and perturbations by effector proteins during effector-triggered susceptibility (ETS). We demonstrate that an immediate increase in actin filament abundance is a conserved and novel component of PTI. Notably, treatment of leaves with a MAMP peptide mimic was sufficient to elicit a rapid change in actin organization in epidermal cells, and this actin response required the host-cell MAMP receptor kinase complex, including FLS2, BAK1 and BIK1. Finally, we found that actin polymerization is necessary for the increase in actin filament density and that blocking this increase with the actin-disrupting drug latrunculin B leads to enhanced susceptibility of host plants to pathogenic and non-pathogenic bacteria.

Arabidopsis myosin XI: a motor rules the tracks.

Plant cell expansion relies on intracellular trafficking of vesicles and macromolecules, which requires myosin motors and a dynamic actin network. Arabidopsis (Arabidopsis thaliana) myosin XI powers the motility of diverse cellular organelles, including endoplasmic reticulum, Golgi, endomembrane vesicles, peroxisomes, and mitochondria. Several recent studies show that there are changes in actin organization and dynamics in myosin xi mutants, indicating that motors influence the molecular tracks they use for transport. However, the mechanism by which actin organization and dynamics are regulated by myosin XI awaits further detailed investigation. Here, using high spatiotemporal imaging of living cells, we quantitatively assessed the architecture and dynamic behavior of cortical actin arrays in a mutant with three Myosin XI (XI-1, XI-2, and XI-K) genes knocked out (xi3KO). In addition to apparent reduction of organ and cell size, the mutant showed less dense and more bundled actin filament arrays in epidermal cells. Furthermore, the overall actin dynamicity was significantly inhibited in the xi3KO mutant. Because cytoskeletal remodeling is contributed mainly by filament assembly/disassembly and translocation/buckling, we also examined the dynamic behavior of individual actin filaments. We found that the xi3KO mutant had significantly decreased actin turnover, with a 2-fold reduction in filament severing frequency. Moreover, quantitative analysis of filament shape change over time revealed that myosin XI generates the force for buckling and straightening of both single actin filaments and actin bundles. Thus, our data provide genetic evidence that three Arabidopsis class XI myosins contribute to actin remodeling by stimulating turnover and generating the force for filament shape change.

Capping protein modulates the dynamic behavior of actin filaments in response to phosphatidic acid in Arabidopsis.

Remodeling of actin filament arrays in response to biotic and abiotic stimuli is thought to require precise control over the generation and availability of filament ends. Heterodimeric capping protein (CP) is an abundant filament capper, and its activity is inhibited by membrane signaling phospholipids in vitro. How exactly CP modulates the properties of filament ends in cells and whether its activity is coordinated by phospholipids in vivo is not well understood. By observing directly the dynamic behavior of individual filament ends in the cortical array of living Arabidopsis thaliana epidermal cells, we dissected the contribution of CP to actin organization and dynamics in response to the signaling phospholipid, phosphatidic acid (PA). Here, we examined three cp knockdown mutants and found that reduced CP levels resulted in more dynamic activity at filament ends, and this significantly enhanced filament-filament annealing and filament elongation from free ends. The cp mutants also exhibited more dense actin filament arrays. Treatment of wild-type cells with exogenous PA phenocopied the actin-based defects in cp mutants, with an increase in the density of filament arrays and enhanced annealing frequency. These cytoskeletal responses to exogenous PA were completely abrogated in cp mutants. Our data provide compelling genetic evidence that the end-capping activity of CP is inhibited by membrane signaling lipids in eukaryotic cells. Specifically, CP acts as a PA biosensor and key transducer of fluxes in membrane signaling phospholipids into changes in actin cytoskeleton dynamics.

Uncovering the molecular interactions underlying MBD2 and MBD3 phase separation.

Chromatin organization controls DNA's accessibility to regulatory factors to influence gene expression. Heterochromatin, or transcriptionally silent chromatin enriched in methylated DNA and methylated histone tails, self-assembles through multivalent interactions with its associated proteins into a condensed, but dynamic state. Liquid-liquid phase separation (LLPS) of key heterochromatin regulators, such as heterochromatin protein 1 (HP1), plays an essential role in heterochromatin assembly and function. Methyl-CpG-binding protein 2 (MeCP2), the most studied member of the methyl-CpG-binding domain (MBD) family of proteins, has been recently shown to undergo LLPS in the absence and presence of methylated DNA. These studies provide a new mechanistic framework for understanding the role of methylated DNA and its readers in heterochromatin formation. However, the details of the molecular interactions by which other MBD family members undergo LLPS to mediate genome organization and transcriptional regulation are not fully understood. Here, we focus on two MBD proteins, MBD2 and MBD3, that have distinct but interdependent roles in gene regulation. Using an integrated computational and experimental approach, we uncover the homotypic and heterotypic interactions governing MBD2 and MBD3 phase separation and DNA's influence on this process. We show that despite sharing the highest sequence identity and structural homology among all the MBD protein family members, MBD2 and MBD3 exhibit differing residue patterns resulting in distinct phase separation mechanisms. Understanding the molecular underpinnings of MBD protein condensation offers insights into the higher-order, LLPS-mediated organization of heterochromatin.

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 IQGAP1's 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 IQGAP1's displacement activity extends to formin-CPz 'decision complexes', promoting end-binding protein turnover at plus-ends. 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.

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, CP). We explore IQGAP1's roles in 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 IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. 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.

Purification of Human Cytoplasmic Actins From Saccharomyces cerevisiae.

Eukaryotic cells rely on actin to support cellular structure, motility, transport, and a wide variety of other cytoplasmic functions and nuclear activities. Humans and other mammals express six closely related isoforms of actin, four of which are found primarily in skeletal, cardiac, and smooth muscle tissues. The final two isoforms, ß and γ, are found in non-muscle cells. Due to the ease of purification, many biochemical studies surveying the functions of actin and its regulators have been carried out with protein purified from skeletal muscle. However, it has become increasingly clear that some activities are isoform specific, necessitating more accessible sources of non-muscle actin isoforms. Recent innovations permit the purification of non-muscle actins from human cell culture and heterologous systems, such as insect cell culture and the yeast Pichia pastoris. However, these systems generate mixtures of actin types or require additional steps to remove purification-related tags. We have developed strains of Saccharomyces cerevisiae (budding yeast) that express single untagged isoforms of either human non-muscle actin (ß or γ) as their sole actin, allowing the purification of individual homogeneous actin isoforms by conventional purification techniques. Key features • Easy growth of yeast as a source of human cytoplasmic actin isoforms. Uses well-established actin purification methods. • The tag-free system requires no post-purification processing.

The formin DAAM1 regulates the deubiquitinase activity of USP10 and integrin homeostasis.

The differentiation of fibroblasts into pathological myofibroblasts during wound healing is characterized by increased cell surface expression of αv-integrins. Our previous studies found that the deubiquitinase (DUB) USP10 removes ubiquitin from αv-integrins, leading to cell surface integrin accumulation, subsequent TGFß1 activation, and pathological myofibroblast differentiation. In this study, a yeast two-hybrid screen revealed a novel binding partner for USP10, the formin, DAAM1. We found that DAAM1 binds to and inhibits USP10's DUB activity through the FH2 domain of DAAM1 independent of its actin functions. The USP10/DAAM1 interaction was also supported by proximity ligation assay (PLA) in primary human corneal fibroblasts. Treatment with TGFß1 significantly increased USP10 and DAAM1 protein expression, PLA signal, and co-localization to actin stress fibers. DAAM1 siRNA knockdown significantly reduced co-precipitation of USP10 and DAAM1 on purified actin stress fibers, and ß1- and ß5-integrin ubiquitination. This resulted in increased αv-, ß1-, and ß5-integrin total protein levels, αv-integrin recycling, and extracellular fibronectin (FN) deposition. Together, our data demonstrate that DAAM1 inhibits USP10's DUB activity on integrins subsequently regulating cell surface αv-integrin localization and FN accumulation.

Visualizing Actin and Microtubule Coupling Dynamics In Vitro by Total Internal Reflection Fluorescence (TIRF) Microscopy.

Traditionally, the actin and microtubule cytoskeletons have been studied as separate entities, restricted to specific cellular regions or processes, and regulated by different suites of binding proteins unique for each polymer. Many studies now demonstrate that the dynamics of both cytoskeletal polymers are intertwined and that this crosstalk is required for most cellular behaviors. A number of proteins involved in actin-microtubule interactions have already been identified (i.e., Tau, MACF, GAS, formins, and more) and are well characterized with regard to either actin or microtubules alone. However, relatively few studies showed assays of actin-microtubule coordination with dynamic versions of both polymers. This may occlude emergent linking mechanisms between actin and microtubules. Here, a total internal reflection fluorescence (TIRF) microscopy-based in vitro reconstitution technique permits the visualization of both actin and microtubule dynamics from the one biochemical reaction. This technique preserves the polymerization dynamics of either actin filament or microtubules individually or in the presence of the other polymer. Commercially available Tau protein is used to demonstrate how actin-microtubule behaviors change in the presence of a classic cytoskeletal crosslinking protein. This method can provide reliable functional and mechanistic insights into how individual regulatory proteins coordinate actin-microtubule dynamics at a resolution of single filaments or higher-order complexes.

Multiple roles for the cytoskeleton in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by more than sixty genes identified through classic linkage analysis and new sequencing methods. Yet no clear mechanism of onset, cure, or effective treatment is known. Popular discourse classifies the proteins encoded from ALS-related genes into four disrupted processes: proteostasis, mitochondrial function and ROS, nucleic acid regulation, and cytoskeletal dynamics. Surprisingly, the mechanisms detailing the contribution of the neuronal cytoskeletal in ALS are the least explored, despite involvement in these cell processes. Eight genes directly regulate properties of cytoskeleton function and are essential for the health and survival of motor neurons, including: TUBA4A, SPAST, KIF5A, DCTN1, NF, PRPH, ALS2, and PFN1. Here we review the properties and studies exploring the contribution of each of these genes to ALS.

Visualizing molecules of functional 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 phosphoinositide lipids, catalyze nucleotide exchange on actin monomers, stimulate formin-mediated actin filament assembly, and bound tubulin dimers (kD = 1.89 µM) to impact microtubule dynamics. In PFN1-deficient mammalian cells, Halo-PFN1 or mApple-PFN1 (mAp-PEN1) restored morphological and cytoskeletal functions. Titrations of self-labeling Halo-ligands 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.

TGFß2 Regulates Human Trabecular Meshwork Cell Contractility via ERK and ROCK Pathways with Distinct Signaling Crosstalk Dependent on the Culture Substrate.

PURPOSE: Transforming growth factor-beta 2 (TGFß2) is a major contributor to the pathologic changes occurring in human trabecular meshwork (HTM) cells in primary open-angle glaucoma (POAG). TGFß2 activates extracellular-signal-regulated kinase (ERK) and Rho-associated kinase (ROCK) signaling pathways, both affecting HTM cell behavior. However, exactly how these signaling pathways converge to regulate HTM cell contractility is unclear. Here, we investigated the molecular mechanism underlying TGFß2-induced pathologic HTM cell contractility, and the crosstalk between ERK and ROCK signaling pathways with different culture substrates. METHODS: Hydrogels were engineered by mixing collagen type I, elastin-like polypeptide, and hyaluronic acid, each containing photoactive functional groups, followed by UV crosslinking. Primary HTM cells were seeded atop pre-formed hydrogels for comparisons with glass, or encapsulated within the hydrogels. Changes in actin cytoskeleton, extracellular matrix (ECM) production, phospho-myosin light chain (p-MLC) levels, and hydrogel contraction were assessed. RESULTS: HTM cell morphology and filamentous (F)-actin organization were affected by the underlying culture substrates. TGFß2 increased HTM cell contractility via ERK and ROCK signaling pathways by differentially regulating F-actin, α-smooth muscle actin (αSMA), fibronectin (FN), and p-MLC in HTM cells. ERK inhibition, even as short as 4 h, further increased TGFß2-induced p-MLC in HTM cells on hydrogels, but not on glass. This translated into hypercontractility of HTM cell-laden hydrogels. ROCK inhibition had precisely the opposite effects and potently relaxed the TGFß2-induced hydrogels. CONCLUSIONS: Our data suggest that ERK signaling negatively regulates ROCK-mediated HTM cell contractility. These findings emphasize the critical importance of using tissue-mimetic ECM substrates for investigating HTM cell physiology and glaucomatous pathophysiology in vitro.

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-poly-L-proline (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.

New twists in actin-microtubule interactions.

Actin filaments and microtubules are cytoskeletal polymers that participate in many vital cell functions including division, morphogenesis, phagocytosis, and motility. Despite the persistent dogma that actin filament and microtubule networks are distinct in localization, structure, and function, a growing body of evidence shows that these elements are choreographed through intricate mechanisms sensitive to either polymer. Many proteins and cellular signals that mediate actin-microtubule interactions have already been identified. However, the impact of these regulators is typically assessed with actin filament or microtubule polymers alone, independent of the other system. Further, unconventional modes and regulators coordinating actin-microtubule interactions are still being discovered. Here we examine several methods of actin-microtubule crosstalk with an emphasis on the molecular links between both polymer systems and their higher-order interactions.

Profilin choreographs actin and microtubules in cells and cancer.

Actin and microtubules play essential roles in aberrant cell processes that define and converge in cancer including: signaling, morphology, motility, and division. Actin and microtubules do not directly interact, however shared regulators coordinate these polymers. While many of the individual proteins important for regulating and choreographing actin and microtubule behaviors have been identified, the way these molecules collaborate or fail in normal or disease contexts is not fully understood. Decades of research focus on Profilin as a signaling molecule, lipid-binding protein, and canonical regulator of actin assembly. Recent reports demonstrate that Profilin also regulates microtubule dynamics and polymerization. Thus, Profilin can coordinate both actin and microtubule polymer systems. Here we reconsider the biochemical and cellular roles for Profilin with a focus on the essential cytoskeletal-based cell processes that go awry in cancer. We also explore how the use of model organisms has helped to elucidate mechanisms that underlie the regulatory essence of Profilin in vivo and in the context of disease.

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge.

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis.

Profilin is an abundant actin monomer-binding protein with critical actin regulatory roles in vivo [1, 2]. However, profilin also influences microtubule dynamics in cells, which may be mediated in part through its interactions with formins that in turn bind microtubules [3, 4]. Specific residues on human profilin-1 (PFN1) are mutated in patients with amyotrophic lateral sclerosis (ALS) [5, 6]. However, the observation that some ALS-linked PFN1 mutants fail to alter cellular actin organization or dynamics [5-8] or in vitro actin-monomer affinity [9] has been perplexing, given that profilin is best understood as an actin regulator. Here, we investigated direct effects of profilin on microtubule dynamics and whether ALS-linked mutations in PFN1 disrupt such functions. We found that human, fly, and yeast profilin homologs all directly enhance microtubule growth rate by several-fold in vitro. Microtubule stimulatory effects were unaffected by mutations in the canonical actin- or poly-proline-binding sites of profilin. Instead, microtubule activities depended on specific surface residues on profilin mutated in ALS patients. Furthermore, microtubule effects were attenuated by increasing concentrations of actin monomers, suggesting competition between actin and microtubules for binding profilin. Consistent with these biochemical observations, a 2-fold increase in the expression level of wild-type PFN1, but not the ALS-linked PFN1 mutants, increased microtubule growth rates in cells. Together, these results demonstrate that profilin directly enhances the growth rate of microtubules. They further suggest that ALS-linked mutations in PFN1 may perturb cellular microtubule dynamics and/or the coordination between the actin and microtubule cytoskeletons, leading to motor neuron degeneration.