Biology subjects

Kamiyama, D.

Publications and source records attributed to Kamiyama, D..

6 recordsLinked to original sources

bioRxiv2026

Distinct postsynaptic morphogenetic strategies across Drosophila embryonic muscles during neuromuscular junction formation

Precise synaptic connectivity emerges through coordinated interactions between neurons and their target cells during development. At the Drosophila embryonic neuromuscular junction (NMJ), postsynaptic muscle fibers actively participate in this process by extending dynamic, actin-rich protrusions termed myopodia that interact with approaching motor growth cones. Previous work focusing on muscle 12 (M12) revealed that myopodia cluster at nascent neuron-muscle contact sites, suggesting that specialized postsynaptic architectures may facilitate synaptic partner selection. However, whether similar morphogenetic strategies operate across the diverse set of embryonic muscles has remained unclear. Here, we establish a genetic imaging toolkit that enables minimally invasive visualization of defined muscle subsets throughout the embryo. Using muscle-specific and stochastic GAL4 drivers to label muscle membranes in vivo, we systematically compare myopodial organization across multiple muscle fibers, including M12, M14, M6, and M7. We find that postsynaptic morphology varies substantially between muscles. M12 displays robust myopodial clustering associated with a prominent sheet-like membrane structure, which we term the muscle lamella, whereas M6 and M14 frequently form myopodial clusters but do not evidently exhibit this structure. In contrast, M7 shows markedly reduced clustering frequency and smaller clusters. These observations reveal previously unrecognized heterogeneity in postsynaptic organization among neighboring muscles during early neuromuscular development. Together, our findings demonstrate that myopodial clustering represents a broadly deployed but differentially organized strategy by which muscles engage motor axons during synaptic partner selection. The imaging toolkit established here provides a foundation for systematic analysis of neuron-muscle interactions across the embryonic musculature and reveals that distinct muscles employ diverse morphogenetic strategies during NMJ assembly.

developmental biology↗

bioRxiv2025

Two-Dimensional Nonlinear Structured Illumination Microscopy with rsEGFP2

Superresolution microscopy enables imaging of subcellular structures and dynamics with nanoscale detail. Among the various superresolution techniques, structured illumination microscopy (SIM) stands out for its compatibility with live-cell imaging. Linear SIM is restricted to a resolution improvement of a factor of two, improving the resolution to about 100 nm. Nonlinear SIM (NSIM) utilizes reversibly switchable fluorescent proteins to generate a nonlinear response, allowing for the collection of higher spatial frequency information and theoretically extending the resolution without limit. By employing rsEGFP2 and patterned depletion illumination (PD) to generate the desired nonlinearity in the fluorescent response, we have successfully achieved 2D PD-NSIM imaging of actin in live U2OS cells with sub-80 nm resolution.

bioengineering↗

bioRxiv2024

Cell-type-specific Labeling of Endogenous Proteins Using the Split GFP System in Drosophila

i.Accurate identification of the locations of endogenous proteins is crucial for understanding their functions in tissues and cells. However, achieving precise cell-type-specific labeling of proteins has been challenging in vivo. A notable solution to this challenge is the self-complementing split green fluorescent protein (GFP1-10/11) system. In this paper, we present a detailed protocol for labeling endogenous proteins in a cell-type-specific manner using the GFP1-10/11 system in fruit flies. This approach depends on the reconstitution of the GFP1-10 and GFP11 fragments, creating a fluorescence signal. We insert the GFP11 fragment into a specific genomic locus while expressing its counterpart, GFP1-10, through an available Gal4 driver line. The unique aspect of this system is that neither GFP1-10 nor GFP11 alone emits fluorescence, enabling the precise detection of protein localization only in Gal4-positive cells expressing the GFP11 tagged endogenous protein. We illustrate this technique using the adhesion molecule gene teneurin-m (Ten-m) as a model, highlighting the generation and validation of GFP11 protein trap lines via Minos-mediated integration cassette (MiMIC) insertion. Furthermore, we demonstrate the cell-type-specific labeling of Ten-m proteins in the larval brains of fruit flies. This method significantly enhances our ability to image endogenous protein localization patterns in a cell-type-specific manner and is adaptable to various model organisms beyond fruit flies.

cell biology↗

bioRxiv2024

Adjacent Neuronal Fascicle Guides Motoneuron 24 Dendritic Branching and Axonal Routing Decisions through Dscam1 Signaling

The formation and precise positioning of axons and dendrites are crucial for the development of neural circuits. Although juxtracrine signaling via cell-cell contact is known to influence these processes, the specific structures and mechanisms regulating neuronal process positioning within the central nervous system (CNS) remain to be fully identified. Our study investigates motoneuron 24 (MN24) in the Drosophila embryonic CNS, which is characterized by a complex yet stereotyped axon projection pattern, known as axonal routing. In this motoneuron, the primary dendritic branches project laterally toward the midline, specifically emerging at the sites where axons turn. We observed that Scp2-positive neurons contribute to the lateral fascicle structure in the ventral nerve cord (VNC) near MN24 dendrites. Notably, the knockout of the Down syndrome cell adhesion molecule (dscam1) results in the loss of dendrites and disruption of proper axonal routing in MN24, while not affecting the formation of the fascicle structure. Through cell-type specific knockdown and rescue experiments of dscam1, we have determined that the interaction between MN24 and Scp2-positive fascicle, mediated by Dscam1, promotes the development of both dendrites and axonal routing. Our findings demonstrate that the holistic configuration of neuronal structures, such as axons and dendrites, within single motoneurons can be governed by local contact with the adjacent neuron fascicle, a novel reference structure for neural circuitry wiring. Significance SummaryWe uncover a key neuronal structure serving as a guiding reference for neural circuitry within the Drosophila embryonic CNS, highlighting the essential role of an adjacent axonal fascicle in precisely coordinating axon and dendrite positioning in motoneuron 24 (MN24). Our investigation of cell-cell interactions between motoneurons and adjacent axonal fascicles-- crucial for initiating dendrite formation, soma migration, and axonal pathfinding in MN24-- emphasizes the neuronal fascicles significance in neural circuit formation through Dscam1-mediated inter-neuronal communication. This enhances our understanding of the molecular underpinnings of motoneuron morphogenesis in Drosophila. Given the occurrence of analogous axon fascicle formations within the vertebrate spinal cord, such structures may play a conserved role in the morphogenesis of motoneurons via Dscam1 across phyla.

neuroscience↗

bioRxiv2022

The Vap33/Eph/Vav/Cdc42 complex confers temporal specification to the outgrowth of primary dendrites in Drosophila neurons

SUMMARYThe formation of primary dendrites (dendritogenesis) significantly affects the overall orientation and coverage of dendritic arborization, limiting the number and types of inputs a neuron can receive. Previously we reported how a Drosophila motoneuron spatially controls the positioning of dendritogenesis through the Dscam1/Dock/Pak1 pathway; however, how the neuron defines the timing of this process remains elusive. Here we show that the Eph receptor tyrosine kinase provides a temporal cue. We find that, at the onset of dendritogenesis, the Eph receptor recruits the Rho Family GEF Vav to the intracellular domain of Eph, which transiently activates the Cdc42 family of small GTPase. We also show that vap33 (vesicle-associated membrane protein-associated protein) mutants exhibit defects in Cdc42 activation and dendritic outgrowth, indicating Vap33 may play an upstream role in Eph signaling. Together, our result and previous studies argue that the formation of primary dendrites requires the proximity of active Cdc42 and membrane-anchored Pak1 driven by collaborative action between two distinct signaling complexes, Vap33/Eph/Vav and Dscam1/Dock. Signal integration from multiple input pathways would represent a general mechanism for the spatiotemporal precision of dendrite branch formation.

neuroscience↗

bioRxiv2018

Autoinhibition of kinesin-1 is essential to the dendrite-specific localization of Golgi outposts

Neuronal polarity relies on the selective localization of cargo to axons or dendrites. The molecular motor kinesin-1 moves cargo into axons but is also active in dendrites. This raises the question of how kinesin-1 activity is regulated to maintain the compartment-specific localization of cargo. Our in vivo structure-function analysis of endogenous Drosophila kinesin-1 reveals a novel role for autoinhibition in enabling the dendrite-specific localization of Golgi outposts. Mutations that disrupt kinesin-1 autoinhibition result in the axonal mislocalization of Golgi outposts. Autoinhibition also regulates kinesin-1 localization. Uninhibited kinesin-1 accumulates in axons and is depleted from dendrites, correlating with the change in outpost distribution and dendrite growth defects. Genetic interaction tests show that a balance of kinesin-1 inhibition and dynein activity is necessary to localize Golgi outposts to dendrites and keep them from entering axons. Our data indicate that kinesin-1 activity is precisely regulated by autoinhibition to achieve the selective localization of dendritic cargo.\n\nSummaryNeuronal polarity relies on the axon-or dendrite-specific localization of cargo by molecular motors such as kinesin-1. These studies show autoinhibition regulates both kinesin-1 activity and localization to keep dendritic cargo from entering axons.

cell biology↗