RESUMO
During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-of-function studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.
Assuntos
Miopatias da Nemalina , Sarcômeros , Animais , Humanos , Sarcômeros/metabolismo , Forminas/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Drosophila/metabolismoRESUMO
Flightless-I is a unique member of the gelsolin superfamily alloying six gelsolin homology domains and leucine-rich repeats. Flightless-I is an established regulator of the actin cytoskeleton, however, its biochemical activities in actin dynamics are still largely elusive. To better understand the biological functioning of Flightless-I we studied the actin activities of Drosophila Flightless-I by in vitro bulk fluorescence spectroscopy and single filament fluorescence microscopy, as well as in vivo genetic approaches. Flightless-I was found to interact with actin and affects actin dynamics in a calcium-independent fashion in vitro. Our work identifies the first three gelsolin homology domains (1-3) of Flightless-I as the main actin-binding site; neither the other three gelsolin homology domains (4-6) nor the leucine-rich repeats bind actin. Flightless-I inhibits polymerization by high-affinity (â¼nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (â¼µM) monomer binding, and does not sever actin filaments. Our work reveals that in the presence of profilin Flightless-I is only able to cap actin filament barbed ends but fails to promote actin assembly. In line with the in vitro data, while gelsolin homology domains 4-6 have no effect on in vivo actin polymerization, overexpression of gelsolin homology domains 1-3 prevents the formation of various types of actin cables in the developing Drosophila egg chambers. We also show that the gelsolin homology domains 4-6 of Flightless-I interact with the C-terminus of Drosophila Disheveled-associated activator of morphogenesis formin and negatively regulates its actin assembly activity.
RESUMO
Disheveled-associated activator of morphogenesis (DAAM) is a diaphanous-related formin protein essential for the regulation of actin cytoskeleton dynamics in diverse biological processes. The conserved formin homology 1 and 2 (FH1-FH2) domains of DAAM catalyze actin nucleation and processively mediate filament elongation. These activities are indirectly regulated by the N- and C-terminal regions flanking the FH1-FH2 domains. Recently, the C-terminal diaphanous-autoregulatory domain (DAD) and the C terminus (CT) of formins have also been shown to regulate actin assembly by directly interacting with actin. Here, to better understand the biological activities of DAAM, we studied the role of DAD-CT regions of Drosophila DAAM in its interaction with actin with in vitro biochemical and in vivo genetic approaches. We found that the DAD-CT region binds actin in vitro and that its main actin-binding element is the CT region, which does not influence actin dynamics on its own. However, we also found that it can tune the nucleating activity and the filament end-interaction properties of DAAM in an FH2 domain-dependent manner. We also demonstrate that DAD-CT makes the FH2 domain more efficient in antagonizing with capping protein. Consistently, in vivo data suggested that the CT region contributes to DAAM-mediated filopodia formation and dynamics in primary neurons. In conclusion, our results demonstrate that the CT region of DAAM plays an important role in actin assembly regulation in a biological context.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Modelos Moleculares , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Pseudópodes/metabolismo , Proteínas de Capeamento de Actina/química , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Substituição de Aminoácidos , Animais , Células Cultivadas , Cristalografia por Raios X , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Embrião não Mamífero/citologia , Deleção de Genes , Glutationa Transferase/química , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/citologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Homologia Estrutural de ProteínaRESUMO
Drosophila melanogaster sarcomere length short (SALS) is a recently identified Wiskott-Aldrich syndrome protein homology 2 (WH2) domain protein involved in skeletal muscle thin filament regulation. SALS was shown to be important for the establishment of the proper length and organization of sarcomeric actin filaments. Here, we present the first detailed characterization of the biochemical activities of the tandem WH2 domains of SALS (SALS-WH2). Our results revealed that SALS-WH2 binds both monomeric and filamentous actin and shifts the monomer-filament equilibrium toward the monomeric actin. In addition, SALS-WH2 can bind to but fails to depolymerize phalloidin- or jasplakinolide-bound actin filaments. These interactions endow SALS-WH2 with the following two major activities in the regulation of actin dynamics: SALS-WH2 sequesters actin monomers into non-polymerizable complexes and enhances actin filament disassembly by severing, which is modulated by tropomyosin. We also show that profilin does not influence the activities of the WH2 domains of SALS in actin dynamics. In conclusion, the tandem WH2 domains of SALS are multifunctional regulators of actin dynamics. Our findings suggest that the activities of the WH2 domains do not reconstitute the presumed biological function of the full-length protein. Consequently, the interactions of the WH2 domains of SALS with actin must be tuned in the cellular context by other modules of the protein and/or sarcomeric components for its proper functioning.