Building Blocks of Functioning Brain: Cytoskeletal Dynamics in Neuronal Development

Neural connectivity requires proper polarization of neurons, guidance to appropriate target locations, and establishment of synaptic connections. From when neurons are born to when they finally reach their synaptic partners, neurons undergo constant rearrangment of the cytoskeleton to achieve appropriate shape and polarity. Of particular importance to neuronal guidance to target locations is the growth cone at the tip of the axon. Growth-cone steering is also dictated by the underlying cytoskeleton. All these changes require spatiotemporal control of the cytoskeletal machinery. This review summarizes the proteins that are involved in modulating the actin and microtubule cytoskeleton during the various stages of neuronal development

Membrane Trafficking in Neuronal Development: Ins and Outs of Neural Connectivity

During development, neurons progress through rapid yet stereotypical shape changes to achieve proper neuronal connectivity. This morphological progression requires carefully orchestrated plasma membrane expansion, insertion of membrane components including receptors for extracellular cues into the plasma membrane and removal and trafficking of membrane materials and proteins to specific locations. This review outlines the cellular machinery of membrane trafficking that play an integral role in neuronal cell shape change and function from initial neurite formation to pathway navigation and synaptogenesis

Recent advances in branching mechanisms underlying neuronal morphogenesis [version 1; referees: 2 approved]

Proper neuronal wiring is central to all bodily functions, sensory perception, cognition, memory, and learning. Establishment of a functional neuronal circuit is a highly regulated and dynamic process involving axonal and dendritic branching and navigation toward appropriate targets and connection partners. This intricate circuitry includes axo-dendritic synapse formation, synaptic connections formed with effector cells, and extensive dendritic arborization that function to receive and transmit mechanical and chemical sensory inputs. Such complexity is primarily achieved by extensive axonal and dendritic branch formation and pruning. Fundamental to neuronal branching are cytoskeletal dynamics and plasma membrane expansion, both of which are regulated via numerous extracellular and intracellular signaling mechanisms and molecules. This review focuses on recent advances in understanding the biology of neuronal branching

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein is a multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity

Ena/VASP regulates mDia2-initiated filopodial length, dynamics, and function

Filopodia are long plasma membrane extensions involved in the formation of adhesive, contractile, and protrusive actin-based structures in spreading and migrating cells. Whether filopodia formed by different molecular mechanisms equally support these cellular functions is unresolved. We used Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP)–deficient MV[superscript D7] fibroblasts, which are also devoid of endogenous mDia2, as a model system to investigate how these different actin regulatory proteins affect filopodia morphology and dynamics independently of one another. Filopodia initiated by either Ena/VASP or mDia2 contained similar molecular inventory but differed significantly in parameters such as number, length, F-actin organization, lifetime, and protrusive persistence. Moreover, in the absence of Ena/VASP, filopodia generated by mDia2 did not support initiation of integrin-dependent signaling cascades required for adhesion and subsequent lamellipodial extension, thereby causing a defect in early cell spreading. Coexpression of VASP with constitutively active mDia2[superscript M/A] rescued these early adhesion defects. We conclude that Ena/VASP and mDia2 support the formation of filopodia with significantly distinct properties and that Ena/VASP regulates mDia2-initiated filopodial morphology, dynamics, and function.National Institutes of Health (U.S.) (Grant GM58801)National Cancer Institute (U.S.). Integrative Cancer Biology Program (Grant 1-U54-CA112967

The Growth Cone Cytoskeleton in Axon Outgrowth and Guidance

Axon outgrowth and guidance to the proper target requires the coordination of filamentous (F)-actin and microtubules (MTs), the dynamic cytoskeletal polymers that promote shape change and locomotion. Over the past two decades, our knowledge of the many guidance cues, receptors, and downstream signaling cascades involved in neuronal outgrowth and guidance has increased dramatically. Less is known, however, about how those cascades of information converge and direct appropriate remodeling and interaction of cytoskeletal polymers, the ultimate effectors of movement and guidance. During development, much of the communication that occurs between environmental guidance cues and the cytoskeleton takes place at the growing tip of the axon, the neuronal growth cone. Several articles on this topic focus on the “input” to the growth cone, the myriad of receptor types, and their corresponding cognate ligands. Others investigate the signaling cascades initiated by receptors and propagated by second messenger pathways (i.e., kinases, phosphatases, GTPases). Ultimately, this plethora of information converges on proteins that associate directly with the actin and microtubule cytoskeletons. The role of these cytoskeletal-associated proteins, as well as the cytoskeleton itself in axon outgrowth and guidance, is the subject of this article

Trim9 Deletion Alters the Morphogenesis of Developing and Adult-Born Hippocampal Neurons and Impairs Spatial Learning and Memory

During hippocampal development, newly born neurons migrate to appropriate destinations, extend axons, and ramify dendritic arbors to establish functional circuitry. These developmental stages are recapitulated in the dentate gyrus of the adult hippocampus, where neurons are continuously generated and subsequently incorporate into existing, local circuitry. Here we demonstrate that the E3 ubiquitin ligase TRIM9 regulates these developmental stages in embryonic and adult-born mouse hippocampal neurons in vitro and in vivo. Embryonic hippocampal and adult-born dentate granule neurons lacking Trim9 exhibit several morphological defects, including excessive dendritic arborization. Although gross anatomy of the hippocampus was not detectably altered by Trim9 deletion, a significant number of Trim9−/− adult-born dentate neurons localized inappropriately. These morphological and localization defects of hippocampal neurons in Trim9−/− mice were associated with extreme deficits in spatial learning and memory, suggesting that TRIM9-directed neuronal morphogenesis may be involved in hippocampal-dependent behaviors

TRIM67 regulates exocytic mode and neuronal morphogenesis via SNAP47

Neuronal morphogenesis involves dramatic plasma membrane expansion, fueled by soluble N-ethylmaleimide-sensitive factor attachment protein eceptors (SNARE)-mediated exocytosis. Distinct fusion modes described at synapses include full-vesicle fusion (FVF) and kiss-and-run fusion (KNR). During FVF, lumenal cargo is secreted and vesicle membrane incorporates into the plasma membrane. During KNR, a transient fusion pore secretes cargo but closes without membrane addition. In contrast, fusion modes are not described in developing neurons. Here, we resolve individual exocytic events in developing murine cortical neurons and use classification tools to identify four distinguishable fusion modes: two FVF-like modes that insert membrane material and two KNR-like modes that do not. Discrete fluorescence profiles suggest distinct behavior of the fusion pore. Simulations and experiments agree that FVF-like exocytosis provides sufficient membrane material for morphogenesis. We find the E3 ubiquitin ligase TRIM67 promotes FVF-like exocytosis in part by limiting incorporation of the Qb/Qc SNARE SNAP47 into SNARE complexes and, thus, SNAP47 involvement in exocytosis

Multi-monoubiquitylation controls VASP-mediated actin dynamics Icon for The Forest of Biologists

The actin cytoskeleton performs multiple cellular functions, and as such, actin polymerization must be tightly regulated. We previously demonstrated that reversible, non-degradative ubiquitylation regulates the function of the actin polymerase VASP in developing neurons. However, the underlying mechanism of how ubiquitylation impacts VASP activity was unknown. Here, we show that mimicking multi-monoubiquitylation of VASP at K240 and K286 negatively regulates VASP interactions with actin. Using in vitro biochemical assays, we demonstrate the reduced ability of multi-monoubiquitylated VASP to bind, bundle, and elongate actin filaments. However, multi-monoubiquitylated VASP maintained the ability to bind and protect barbed ends from capping protein. Finally, we demonstrate the electroporation of recombinant multi-monoubiquitylated VASP protein altered cell spreading morphology. Collectively, these results suggest a mechanism in which ubiquitylation controls VASP-mediated actin dynamics

TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

Extracellular netrin-1 and its receptor deleted in colorectal cancer (DCC) promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that tripartite motif protein 9 (TRIM9)-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased soluble N-ethylmaleimide attachment protein receptor (SNARE)-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons