A two-step actin polymerization mechanism drives dendrite branching

Abstract

Background Dendrite morphogenesis plays an essential role in establishing the connectivity and receptive fields of neurons during the development of the nervous system. To generate the diverse morphologies of branched dendrites, neurons use external cues and cell surface receptors to coordinate intracellular cytoskeletal organization; however, the molecular mechanisms of how this signaling forms branched dendrites are not fully understood. Methods We performed in vivo time-lapse imaging of the PVD neuron in C. elegans in several mutants of actin regulatory proteins, such as the WAVE Regulatory Complex (WRC) and UNC-34 (homolog of Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP)). We examined the direct interaction between the WRC and UNC-34 and analyzed the localization of UNC-34 in vivo using transgenic worms expressing UNC-34 fused to GFP. Results We identify a stereotyped sequence of morphological events during dendrite outgrowth in the PVD neuron in C. elegans. Specifically, local increases in width (“swellings”) give rise to filopodia to facilitate a “rapid growth and pause” mode of growth. In unc-34 mutants, filopodia fail to form but swellings are intact. In WRC mutants, dendrite growth is largely absent, resulting from a lack of both swelling and filopodia formation. We also found that UNC-34 can directly bind to the WRC. Disrupting this binding by deleting the UNC-34 EVH1 domain prevented UNC-34 from localizing to swellings and dendrite tips, resulting in a stunted dendritic arbor and reduced filopodia outgrowth. Conclusions We propose that regulators of branched and linear F-actin cooperate to establish dendritic branches. By combining our work with existing literature, we propose that the dendrite guidance receptor DMA-1 recruits the WRC, which polymerizes branched F-actin to generate “swellings” on a mother dendrite. Then, WRC recruits the actin elongation factor UNC-34/Ena/VASP to initiate growth of a new dendritic branch from the swelling, with the help of the actin-binding protein UNC-115/abLIM. Extension of existing dendrites also proceeds via swelling formation at the dendrite tip followed by UNC-34-mediated outgrowth. Following dendrite initiation and extension, the stabilization of branches by guidance receptors further recruits WRC, resulting in an iterative process to build a complex dendritic arbor.

The intrinsically disordered cytoplasmic tail of a dendrite branching receptor uses two distinct mechanisms to regulate the actin cytoskeleton

“…HPO-30, by binding to actin filaments and the WRC at the same locations on the membrane, would promote the formation of more highly branched actin networks, likely in a manner analogous to CapZ ( Figure 6 , bottom). This dual actin binding ability could be one mechanism by which the swellings at branch points observed in PVD neurons are formed (Shi et al, 2021). Further investigation into how the HPO-30 ICD binds to both the WRC and actin will potentially reveal a new class of proteins that can regulate multiple steps of the actin polymerization process, allowing for greater control of local actin dynamics.…”

“…This interaction recruits the WRC to the membrane and simultaneously promotes WRC activation by Rac1, which in turn stimulates Arp2/3 to produce branched actin filaments. In (4), the dual actions of HPO-30 ICD cooperatively promote the formation of branched actin networks, which can cause the "swelling" of dendrite observed in previous studies (Shi et al, 2021), an important prerequisite for the outgrowth of a new dendrite branch.…”

“…These two distinct functions by the same membrane receptor provide an exciting model to explain how HPO-30 regulates local actin dynamics to facilitate dendrite branching ( Figure 7 ). Synergistic action of both functions of HPO-30 would promote the formation of highly branched actin networks, giving rise to dendritic “swellings” observed in PVD neurons prior to dendrite branch outgrowth ( Figure 7 , step 4) (Shi et al, 2021).…”

“…GST pull-down assays were performed as previously described (Shi et al, 2021). Briefly, 20 µL of GSH-Sepharose beads were mixed with bait protein and prey protein in 1 mL of pull-down buffer (50 mM NaCl, 10 mM HEPES pH 7.0, 5% (w/v) glycerol, 5 mM 2- mercaptoethanol, and 0.05% Triton X-100).…”

Nascent dendrite branches initiated by a localized burst of Spire-dependent actin polymerization

“…Dendrite filopodia are structurally distinct from conventional filopodia, with an unusual network-like cytoskeletal organization characterized by both branched and linear filaments of mixed polarity (Korobova and Svitkina, 2010), suggesting involvement of multiple actin regulatory proteins with distinct activities. Few studies have identified specific actin regulators involved in forming new branches (Hou et al, 2015; Nithianandam and Chien, 2018; Shi et al, 2021; Sturner et al, 2022; Sturner et al, 2019; Zou et al, 2018), and none have pinpointed their regulation of actin to the location and timing of nascent branch outgrowth. In sensory neurons of living Drosophila larvae, the locations of dendrite filopodia initiation sites are pre-figured by patches of actin polymer (Andersen et al, 2005; Nithianandam and Chien, 2018; Sturner et al ., 2019), as has been proposed for dendrite filopodia in the mouse brain also (Willig et al, 2014).…”