Multicomponent depolymerization of actin filament pointed ends by cofilin and cyclase-associated protein depends upon filament age.

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by "aging" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus "treadmill" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-P i actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-P i filaments. Interestingly, the maximal rates of ADP-P i filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-P i pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of P i release in the aging process.

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 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 underlying the neural phenotypes associated with GM3SD are unknown. 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 cells carrying either variant, 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. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in 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 erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

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

Cells control actin assembly by regulating reactions at actin filament barbed ends. Formins accelerate elongation, capping protein (CP) arrests growth and twinfilin promotes depolymerization at barbed ends. How these distinct activities get integrated within a shared cytoplasm is unclear. Using microfluidics-assisted TIRF microscopy, we find that formin, CP and twinfilin can simultaneously bind filament barbed ends. Three­color, single-molecule experiments reveal that twinfilin cannot bind barbed ends occupied by formin unless CP is present. This trimeric complex is short-lived (~1 s), and results in dissociation of CP by twinfilin, promoting formin-based elongation. Thus, the depolymerase twinfilin acts as a pro-formin pro-polymerization factor when both CP and formin are present. While one twinfilin binding event is sufficient to displace CP from the barbed-end trimeric complex, ~31 twinfilin binding events are required to remove CP from a CP-capped barbed end. Our findings establish a paradigm where polymerases, depolymerases and cappers together tune actin assembly.

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.

Pointed-end processive elongation of actin filaments by Vibrio effectors VopF and VopL.

According to the cellular actin dynamics paradigm, filaments grow at their barbed ends and depolymerize predominantly from their pointed ends to form polar structures and do productive work. We show that actin can elongate at the pointed end when assisted by Vibrio VopF/L toxins, which act as processive polymerases. In cells, processively moving VopF/L speckles are inhibited by factors blocking the pointed but not barbed ends. Multispectral single-molecule imaging confirmed that VopF molecules associate with the pointed end, actively promoting its elongation even in the presence of profilin. Consequently, VopF/L can break the actin cytoskeleton's polarity by compromising actin-based cellular processes. Therefore, actin filament design allows processive growth at both ends, which suggests unforeseen possibilities for cellular actin organization, particularly in specialized cells and compartments.

Derivation of Peripheral Nociceptive, Mechanoreceptive, and Proprioceptive Sensory Neurons from the same Culture of Human Pluripotent Stem Cells.

The three peripheral sensory neuron (SN) subtypes, nociceptors, mechanoreceptors, and proprioceptors, localize to dorsal root ganglia and convey sensations such as pain, temperature, pressure, and limb movement/position. Despite previous reports, to date no protocol is available allowing the generation of all three SN subtypes at high efficiency and purity from human pluripotent stem cells (hPSCs). We describe a chemically defined differentiation protocol that generates all three SN subtypes from the same starting population, as well as methods to enrich for each individual subtype. The protocol yields high efficiency and purity cultures that are electrically active and respond to specific stimuli. We describe their molecular character and maturity stage and provide evidence for their use as an axotomy model; we show disease phenotypes in hPSCs derived from patients with familial dysautonomia. Our protocol will allow the modeling of human disorders affecting SNs, the search for treatments, and the study of human development.