Biology subjects

Vizcarra, C. L.

Publications and source records attributed to Vizcarra, C. L..

5 recordsLinked to original sources

bioRxiv2022

Metallothionein-3 attenuates the effect of Cu2+ ions on actin filaments

Metallothionein 3 (MT-3) is a cysteine-rich metal-binding protein that is expressed in the mammalian central nervous system and kidney. Various reports have posited a role for MT-3 in regulating the actin cytoskeleton by promoting the assembly of actin filaments. We generated purified, recombinant mouse MT-3 of known metal compositions, either with zinc (Zn), lead (Pb), or copper/zinc (Cu/Zn) bound. None of these forms of MT-3 accelerated actin filament polymerization in vitro, either with or without the actin binding protein profilin. Furthermore, using a co-sedimentation assay, we did not observe Zn-bound MT-3 in complex with actin filaments. Cu2+ ions on their own induced rapid actin polymerization, an effect that we attribute to filament fragmentation. This effect of Cu2+ is reversed by adding either EGTA or Zn-bound MT-3, indicating that either molecule can chelate Cu2+ from actin. Altogether, our data indicate that recombinant MT-3 does not directly bind actin but it does attenuate the Cu-induced fragmentation of actin filaments.

biochemistry↗

bioRxiv2022

Alterations to the broad-spectrum formin inhibitor SMIFH2 improve potency

SMIFH2 is a small molecule inhibitor of the formin family of cytoskeletal regulators that was originally identified in a screen for suppression of actin polymerization induced by the mouse formin Diaphanous 1 (mDia1). Despite widespread use of this compound, it is unknown whether SMIFH2 inhibits all human formins. Additionally, the nature of protein/inhibitor interactions remains elusive. We assayed SMIFH2 against human formins representing six of the seven mammalian classes and found inhibitory activity against all formins tested. We synthesized a panel of SMIFH2 derivatives and found that, while many alterations disrupt SMIFH2 activity, substitution of an electron-donating methoxy group in place of the bromine along with halogenation of the furan ring increases potency by approximately five-fold. Similar to SMIFH2, the active derivatives are also pan-inhibitors for the formins tested. This result suggests that while potency can be improved, the goal of distinguishing between highly conserved FH2 domains may not be achievable using the SMIFH2 scaffold.

biochemistry↗

bioRxiv2021

Variable autoinhibition among deafness-associated variants of Diaphanous 1 (DIAPH1)

One of the earliest mapped human deafness genes, DIAPH1, encodes the formin DIAPH1. To date, at least three distinct mutations in the C-terminal domains and two additional mutations in the N-terminal region are associated with autosomal dominant hearing loss. The underlying molecular mechanisms are not known, and the role of formins in the inner ear is not well understood. In this study we use biochemical assays to test the hypotheses that autoinhibition and/or actin assembly activities are disrupted by DFNA1 mutations. Our results indicate that C-terminal mutant forms of DIAPH1 are functional in vitro and promote actin filament assembly. Similarly, N-terminal mutants are well-folded and have quaternary structures and thermal stabilities similar to the WT protein. The strength of the autoinhibitory interactions varies widely among mutants, with the ttaa, A265S and I530S mutations having an affinity similar to WT and the 1213x and {Delta}ag mutations completely abolishing autoinhibition. These data indicate that, in some cases, hearing loss may be linked to reduced inhibition of actin assembly.

biochemistry↗

bioRxiv2019

Spire stimulates nucleation by Cappuccino and binds both ends of actin filaments

An actin mesh fills both mouse and fly oocytes. The meshes are built by a conserved mechanism and used to establish polarity. Two actin nucleators, Spire and Cappuccino, collaborate to build actin filaments that connect vesicles and the cortex. Direct interaction between Spire and Cappuccino is required for in vitro synergistic actin assembly; however, we understand little about why the interaction is necessary. To mimic the geometry of Spire and Cappuccino in vivo, we immobilized Spire on beads. We found that increased nucleation is a major part of synergy and that Spire alone binds both barbed- and pointed-ends of actin filaments. We identified Spires barbed-end binding domain. Partial rescue of fertility by a loss-of-function mutant indicates that barbed-end binding is not necessary for Spires in vivo function, but that it may play a role under normal circumstances. We propose that Spire stimulates nucleation by Cappuccino in a manner similar to the collaboration between APC and mDia1.\n\nSummaryActin nucleators Cappuccino and Spire collaborate to build an actin mesh in oocytes. Data demonstrate that the collaboration leads to synergistic actin nucleation, as opposed to elongation. Further, Spire binds both ends of polar, actin filaments, resolving a long-outstanding question.

biochemistry↗

bioRxiv2017

The Neuron Specific Formin Delphilin Nucleates Actin Filaments but Does Not Enhance Elongation

The formin Delphilin binds the glutamate receptor, GluR{delta}2, in dendritic spines of Purkinje cells. Both proteins play a role in learning. To understand how Delphilin functions in neurons, we studied the actin assembly properties of this formin. Formins have a conserved formin homology 2 domain, which nucleates and remains associated with the fast growing end of actin filaments, influencing filament growth with input from the adjacent formin homology 1 domain. The strength of nucleation and elongation varies widely across formins. Additionally, most formins have conserved domains that regulate actin assembly through an intramolecular interaction. Delphilin is distinct from other formins in several ways: its expression is limited to Purkinje cells; it lacks autoinhibitory domains; its formin homology 1 domains has minimal proline-rich sequence. We found that Delphilin is an actin nucleator that does not accelerate elongation, although it binds tightly to the barbed end of filaments. In addition, Delphilin exhibits a preference for actin isoforms, which has not been described or systematically studied in other formins. Finally, Delphilin is the first formin studied that is not regulated by intramolecular interactions. We speculate how the activity we observe is consistent with its localization in the small dendritic spines.\n\nAbbreviations: FH1, formin homology 1; FH2, formin homology 2; DID, diaphanous inhibitory domain; DAD, diaphanous autoinhibitory domain; LTD, long term depression; PSD, post synaptic density; PDZ, post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (Dlg1), and zonula occludens-1 protein (zo-1).

biochemistry↗