The overall goal of this application is to clarify the molecular organization and function of enigmatic axonal actin assemblies that my lab recently discovered. Actin organization along axon shafts has been relatively ignored for decades, probably because routine staining ? using the classic actin-filament marker phalloidin ? only shows a patchy, uninteresting pattern. However, recent studies from us and others' using super-resolution microscopy and low-light imaging have revealed a hidden world of actin in axon shafts ? replete with elaborate circumferential rings underneath the plasma membrane, and rapidly elongating linear filaments along the axis of the axon shaft. We have been fortunate to be at the forefront of these discoveries, and our studies have revealed fascinating actin dynamics in axons. Specifically, we found that axons have focal ?hotspots? of actin ? spaced ~ 3-4 m apart ? where actin continuously polymerizes and depolymerizes. These hotspots give rise to long actin filaments that rapidly (and bidirectionally) elongate along the axon shaft (we named these ?actin trails?). Actin rings, trails and hotspots are seen in axons in vivo, and we will use in vivo model systems in this proposal as well. Although the overall assemblies have been seen, molecular events underlying the generation and maintenance of these structures is unclear. Importantly, their functional roles in axons are unknown. Here we propose three specific aims to clarify the organization and function of these newly-discovered actin assemblies:
Aim #1 : Identify mechanisms initiating actin trails in axons.
Aim #2 : Identify mechanisms elongating actin trails in axons.
Aim #3 : Determine functions of axonal actin assemblies.

Public Health Relevance

Actin is a highly conserved protein and an integral component of the cytoskeleton ? involved in numerous physiologic activities, as well as in many neurodevelopmental and neurodegenerative diseases; particularly autism. Using probes that selectively report actin filaments and advanced microscopy techniques that allow visualization of sub-diffraction structures, we discovered previously unknown actin assemblies along the shafts of axons. The goal of the proposed experiments is to dissect the molecular mechanisms underlying these axonal actin assemblies, and also learn what their functions are.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Special Emphasis Panel (ZRG1)
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Miller, Daniel L
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University of California, San Diego
Schools of Medicine
La Jolla
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Dubey, Pankaj; Jorgenson, Kent; Roy, Subhojit (2018) Actin Assemblies in the Axon Shaft - some Open Questions. Curr Opin Neurobiol 51:163-167
Sun, Jichao; Roy, Subhojit (2018) The physical approximation of APP and BACE-1: A key event in alzheimer's disease pathogenesis. Dev Neurobiol 78:340-347
Leterrier, Christophe; Dubey, Pankaj; Roy, Subhojit (2017) The nano-architecture of the axonal cytoskeleton. Nat Rev Neurosci 18:713-726
Ganguly, Archan; Han, Xuemei; Das, Utpal et al. (2017) Hsc70 chaperone activity is required for the cytosolic slow axonal transport of synapsin. J Cell Biol 216:2059-2074
Das, Utpal; Wang, Lina; Ganguly, Archan et al. (2016) Visualizing APP and BACE-1 approximation in neurons yields insight into the amyloidogenic pathway. Nat Neurosci 19:55-64
Ladt, Kelsey; Ganguly, Archan; Roy, Subhojit (2016) Axonal actin in action: Imaging actin dynamics in neurons. Methods Cell Biol 131:91-106
Roy, Subhojit (2016) Waves, rings, and trails: The scenic landscape of axonal actin. J Cell Biol 212:131-4
Ganguly, Archan; Tang, Yong; Wang, Lina et al. (2015) A dynamic formin-dependent deep F-actin network in axons. J Cell Biol 210:401-17
Ganguly, Archan; Roy, Subhojit (2014) Using photoactivatable GFP to track axonal transport kinetics. Methods Mol Biol 1148:203-15
Roy, Subhojit (2014) Seeing the unseen: the hidden world of slow axonal transport. Neuroscientist 20:71-81

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