Specifically, several families of motor proteins, which carry transport vesicles along microtubule and actin tracks, have been described ( Hirokawa and Takemura, 2005 Kneussel and Wagner, 2013). Many aspects of axonal and dendritic transport are well characterized ( Hirokawa, 1993 Maday et al., 2014 Roy, 2014 Twelvetrees, 2020). Indeed, defects in axonal transport have been implicated in a variety of neurodegenerative diseases ( Hung and Link, 2011 Maday et al., 2014 May-simera and Liu, 2013 Vicario-Orri et al., 2014). How this molecular and cellular polarity is maintained, specifically in the case of highly extended axons, is an essential question since preserving this extreme polarity underlies neuronal function. For example, in chemical synapses, dendrites require a steady supply of receptors and proteins that are involved in postsynaptic signaling, whereas axons require the machinery that drives the synaptic vesicle (SV) cycle, including the exocytosis of neurotransmitters.
Within this polarized framework, axons and dendrites are highly adapted to carry out different functions and, consequently, each harbor somewhat distinct molecular constituents. These highly specialized and asymmetric cells form elaborate axonal and dendritic arbors, with some axons extending great distances (e.g., axons in a blue whale can reach a length of 30 meters Smith, 2009).
Neurons present a dramatic example of cell polarization. This study will be of interest to cell biologists and neuroscientists alike because (i) it provides a major advance in our understanding of nerve cell development and function, (ii) it demonstrates the usefulness of the RUSH approach in nerve cell biology, and (iii) it stresses the importance of tight control of reporter (over)expression, which is important in many other contexts. The corresponding evidence is compelling, and, furthermore, the authors' observation that even slightly excessive expression levels of the fluorescently tagged reporters occlude the specific axonal trafficking so that proteins distribute indiscriminately into axons and dendrites, explains why previous studies often failed to detect specific axonal trafficking of synaptic vesicle proteins. By using a very-low-level expression paradigm to express fluorescently tagged reporter proteins in neurons, a method to allow their triggered and 'synchronous' exit from the endoplasmic reticulum (RUSH), and live cell imaging, the authors describe a specific axonal trafficking pathway for the synaptic vesicle proteins Synaptotagmin-1 and Synaptobrevin-2. how these highly polarized cells achieve the specific and differential distribution of proteins and organelles into their axonal and dendritic compartments – the study is an important step forward in this context. The authors explored a key question in nerve cell biology, i.e. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels.
Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons.
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