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Published on: July 30, 2014
Profilin Directly Promotes Microtubule Growth through Residues Mutated in Amyotrophic Lateral Sclerosis
Jessica L Henty-Ridilla1, M Angeles Juanes1, Bruce L Goode1
1Department of Biology, Brandeis University, 415 South Street, Waltham, MA 02454, USA.
Abstract:
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.
Insights
Profilin directly enhances microtubule growth. ALS-linked mutations in profilin-1 (PFN1) may disrupt microtubule dynamics and actin-microtubule coordination, potentially contributing to motor neuron degeneration.
Area of Science:
- Cell Biology
- Biochemistry
- Neuroscience
Background:
- Profilin is a key actin-binding protein involved in regulating actin dynamics.
- Profilin's influence on microtubule dynamics and its role in amyotrophic lateral sclerosis (ALS) are not fully understood.
- Some ALS-linked mutations in profilin-1 (PFN1) do not affect actin binding or cellular actin organization, posing a paradox.
Purpose of the Study:
- To investigate the direct effects of profilin on microtubule dynamics.
- To determine if ALS-linked mutations in PFN1 disrupt profilin's functions related to microtubules.
- To explore the interplay between actin and microtubule regulation by profilin.
Main Methods:
- In vitro biochemical assays to measure microtubule growth rates in the presence of profilin.
- Site-directed mutagenesis of PFN1 to create ALS-linked variants.
- Cellular experiments measuring microtubule growth rates with altered PFN1 expression levels.
Main Results:
- Profilin homologs from human, fly, and yeast directly and significantly enhance microtubule growth rates in vitro.
- Microtubule stimulation by profilin is independent of its canonical actin- or poly-proline-binding sites.
- ALS-linked PFN1 mutations, but not mutations in actin-binding sites, affect profilin's interaction with microtubules.
- Increased actin monomer concentration attenuates profilin's microtubule-promoting activity, suggesting competition.
- Increased expression of wild-type PFN1, but not ALS mutants, enhances microtubule growth in cells.
Conclusions:
- Profilin directly promotes microtubule polymerization.
- ALS-linked PFN1 mutations can impair profilin's ability to enhance microtubule growth.
- Dysregulation of microtubule dynamics and/or actin-microtubule coordination by mutant PFN1 may contribute to ALS pathogenesis.
- Profilin acts as a crucial regulator linking actin and microtubule cytoskeletal networks.
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