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The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

ABSTRACT: 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 (?nM) filament barbed end capping, moderately facilitates nucleation by low-affinity (??M) 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.

SUBMITTER: Pinter R

PROVIDER: S-EPMC7509490 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

Pintér Réka R Huber Tamás T Bukovics Péter P Gaszler Péter P Vig Andrea Teréz AT Tóth Mónika Ágnes MÁ Gazsó-Gerhát Gabriella G Farkas Dávid D Migh Ede E Mihály József J Bugyi Beáta B

Frontiers in molecular biosciences 20200908

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 <i>Drosophila</i> Flightless-I by <i>in vitro</i> bulk fluorescence spectroscopy and single filament fluorescence microscopy, a ...[more]

PMID: 33033719

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Project description:Background & aimsCell stress signaling pathways result in phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1 or EIF2A), which affects regulation of protein translation. Translation reprogramming mitigates stress by activating pathways that result in autophagy and cell death, to eliminate damaged cells. Actin is modified during stress and EIF2A is dephosphorylated to restore homeostasis. It is not clear how actin affects EIF2A signaling. We studied the actin-binding proteins villin 1 (VIL1) and gelsolin (GSN) in intestinal epithelial cells (IECs) to determine whether they respond to cell stress response and affect signaling pathways.MethodsWe performed studies with mice with disruptions in Vil1 and Gsn (double-knockout mice). Wild-type (WT) mice either were or were not (controls) exposed to cell stressors such as tumor necrosis factor and adherent-invasive Escherichia coli. Distal ileum tissues were collected from mice; IECs and enteroids were cultured and analyzed by histology, immunoblots, phalloidin staining, immunohistochemistry, electron microscopy, and flow cytometry. HT-29 cells were incubated with cell stressors such as DTT, IFN, and adherent-invasive E coli or control agents; cells were analyzed by immunoblots and quantitative polymerase chain reaction. Green fluorescent protein and green fluorescent protein tagged mutant EIF2A were expressed from a lentiviral vector. The mouse immunity-related GTPase (IRGM1) was overexpressed in embryonic fibroblasts from dynamin1 like (DNM1L) protein-knockout mice or their WT littermates. IRGM1 was overexpressed in embryonic fibroblasts from receptor interacting serine/threonine kinase 1-knockout mice or their WT littermates. Human IRGM was overexpressed in human epithelial cell lines incubated with the DNM1L-specific inhibitor Mdivi-1. Mitochondria were analyzed by semi-quantitative confocal imaging. We performed immunohistochemical analyses of distal ileum tissues from 6-8 patients with Crohn's disease (CD) and 6-8 individuals without CD (controls).ResultsIn IECs exposed to cell stressors, EIF2A signaling reduced expression of VIL1 and GSN. However, VIL1 and GSN were required for dephosphorylation of EIF2A and recovery from cell stress. In mouse and human IECs, prolonged, unresolved stress was accompanied by continued down-regulation of VIL1 and GSN, resulting in constitutive phosphorylation of EIF2A and overexpression of IRGM1 (or IRGM), which regulates autophagy. Overexpression of IRGM1 (or IRGM) induced cell death by necroptosis, accompanied by release of damage-associated molecular patterns (DAMPs). In double-knockout mice, constitutive phosphorylation of EIF2A and over-expression of IRGM1 resulted in spontaneous ileitis that resembled human CD in symptoms and histology. Distal ileum tissues from patients with CD had lower levels of VIL1 and GSN, increased phosphorylation of EIF2A, increased levels of IRGM and necroptosis, and increased release of nuclear DAMPs compared with controls.ConclusionsIn studies of intestinal epithelial tissues from patients with CD and embryonic fibroblasts from mice, along with enteroids and human IEC lines, we found that induction of cell stress alters the cytoskeleton in IECs via changes in the actin-binding proteins VIL1 and GSN. Acute changes in actin dynamics increase IEC survival, whereas long-term changes in actin dynamics lead to IEC death and intestinal inflammation. IRGM regulates necroptosis and release of DAMPs to induce gastrointestinal inflammation, linking IRGM activity with CD.

Dataset Information

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

Publications

The Activities of the Gelsolin Homology Domains of Flightless-I in Actin Dynamics.

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