Ulrichs, H. | bioRxiv Science

bioRxiv2023

Multicomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein

Living cells assemble their actin networks by regulating reactions at the barbed end of actin filaments. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How cells integrate these disparate activities within a shared cytoplasm to produce diverse actin networks, each with distinct morphologies and finely tuned assembly kinetics, is unclear. We used microfluidics-assisted TIRF microscopy to investigate how formin mDia1, CP and twinfilin influence the elongation of actin filament barbed ends. We discovered that the three proteins can simultaneously bind a barbed end in a multiprotein complex. Three-color single molecule experiments showed that twinfilin cannot bind actin filament ends occupied by formin mDia1 unless CP is present. The trimeric complex is short-lived ([~]1s) and results in rapid dissociation of CP by twinfilin causing resumption of rapid formin- based elongation. Thus, the depolymerase twinfilin acts as a pro-formin factor that promotes polymerization when both CP and formin are present. While a single twinfilin binding event is sufficient to displace CP from the trimeric complex, it takes about 30 independent twinfilin binding events to remove capping protein from CP-bound barbed end. Our findings establish a new paradigm in which polymerases, depolymerases and cappers work in concert to tune cellular actin assembly.

biophysics↗

bioRxiv2021

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 Synthase Deficiency variants

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single 3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.

neuroscience↗