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

Abstract

SUMMARY 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. Further, 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 two-fold increase in the expression level of wildtype 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. Further, they 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.

Profilin as a dual regulator of actin and microtubule dynamics

“…As referred to above, the effect of Pfn1 in promoting microtubule growth rates in vitro and in neuroblastoma cells contrast with data obtained in melanoma cells (Nejedla et al, ). Surprisingly, when Pfn1 was depleted from N2A cells, an increased microtubule growth speed was also obtained, although to a smaller extent than that produced by Pfn1 overexpression (Henty‐Ridilla et al, ). It was therefore concluded that microtubule growth rates are probably highly sensitive to Pfn1 levels in either direction, although no mechanism has been put forward to explain this apparent discrepancy.…”

“…To test the biological relevance of the observations made in vitro, Pfn1 was overexpressed in mouse neuroblastoma (N2A) cells. By tracking microtubule (+)‐ends, microtubule growth speed was increased by almost threefold and a significant increase in microtubule entry into filopodia was also observed (Henty‐Ridilla et al, ). As referred to above, the effect of Pfn1 in promoting microtubule growth rates in vitro and in neuroblastoma cells contrast with data obtained in melanoma cells (Nejedla et al, ).…”

“…Using TIRF microscopy, two of the initial ALS mutants identified (M114T and G118V) totally failed to bind microtubules and to enhance microtubule growth, suggesting that their inability to promote microtubule growth speed was dependent on their affinity to microtubules. Interestingly, the effect of profilin on microtubule dynamics, was decreased by increasing the concentration of actin monomers (Henty‐Ridilla et al, ). This suggested a competition between actin and microtubules for profilin binding, agreeing well with the proximity of the microtubule and actin‐binding sites on the molecule.…”

“…This suggested a competition between actin and microtubules for profilin binding, agreeing well with the proximity of the microtubule and actin‐binding sites on the molecule. Moreover, these findings suggest that ALS‐linked mutations in Pfn1 alter microtubule dynamics and/or the actin‐microtubule interplay, ultimately leading to motor neuron degeneration (Henty‐Ridilla et al, ). Additionally, one can expect that other profilin variants may show a similar capacity to bind to microtubules as the amino acids recently identified as possible microtubule‐binding residues (M114 and G118) are conserved.…”

“…As such, although originally identified as actin sequestering proteins, profilins have been characterized as key molecular regulators of actin polymerization dynamics that provide a connection between membrane lipids and cytoskeleton components. In recent years, Pfn1 was shown to contain specific residues enabling its direct interaction with microtubules (Henty‐Ridilla, Juanes, & Goode, ) (Figure a). This finding powered the complexity and importance of profilins as proteins capable of interacting with multiple cytoskeleton components.…”