Krishanu Ray

The GTPase Rab4, which is essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed transient contraction of gross synapse density in the ventral neuropil region at a specific larval stage. This reduction in the gross synapse density coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruits a PX-domain-containing motor protein, Klp98A, accelerating their synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via Vps34-depedent regulation of directed axonal transport of endosomes.

Sensory cilia have a complex bipartite architecture containing 9+0 connecting cilia at the inner segment and singlet microtubule-supported highly membranous outer segments essential for receptor display. The mechanisms underlying the formation of such highly branched morphology and its microtubule-rich ciliary cytoskeleton are unclear. Here, we show that individual olfactory cilium inside the large basiconic sensillum grows in episodic steps following several pulsatile influxes of tubulin in developing Drosophila antenna. Transient elevations of the microtubule end-binding protein EB1 precede the tubulin influx events. We also demonstrate that EB1 interacts explicitly with the cargo-binding tail domain of Drosophila KLP68D, a kinesin-2β orthologue. Loss of EB1 in olfactory neurons during the outer segment growth reduces the tubulin influx and affects cilia stability. Finally, we show that the EB1 and tubulin influxes into the distal outer segment of the olfactory cilia require kinesin-2. Altogether, our findings elucidate a role of active EB1 transport in promoting the growth and stability of long-lived metazoan cilia involved in sensory perception.

Rab4 GTPase, essential for endosomal sorting and trafficking, is implicated in synaptic atrophy and dementia. To uncover the underlying mechanism, we studied the correlation between Rab4 vesicle transport in axons and episodic remodeling of synapses in the central nervous system (CNS) of Drosophila larvae. We found that synapse-bound traffic and presynaptic enrichment of Rab4 vesicles increase during the programmed, transient contraction of synapses in the ventral neuropil region at a specific larval stage. This coincides with the episodic activation of insulin and Vps34-mediated signaling, which elevates phosphatidylinositol-3-phosphate levels on Rab4 vesicles. The presence of this phospholipid on Rab4-associated vesicles recruit a PX-domain-containing motor protein, Klp98A, accelerating synapse-directed traffic. This, in turn, increases presynaptic enrichment of Rab4 during the developmentally programmed synapse contraction phase. Our findings elucidate the molecular mechanism that regulates developmental synaptic plasticity in the CNS via insulin signaling and directed axonal transport of endosomes. Author summary: Healthy brain function relies on the growth and remodeling of synaptic connections between nerve cells. This process involves carefully orchestrated movements of proteins and membranes inside the neurites. For instance, endosome movement in the nerve cell extensions helps guide neuronal growth, recycle parts of the synapse, and alter signal transmissions between neurons. Rab GTPases, a group of small G-proteins, are crucial for these processes. Here, we aimed to understand what triggers these processes and how the ’supply chain’ within nerve cell extensions is connected to changes at the synapse. In fruit fly larvae, we observed that the movement of Rab4 vesicles in the nerve cell extensions is inversely related to gross synapse content. A further examination revealed that onset of insulin signaling accelerates the Rab4 vesicles towards synapse through a lipid kinase called Vps34, which makes a signaling lipid called phospatidylinnositol-3-phosphate. Higher levels this lipid molecule on Rab4 vesicles attract a motor protein, Klp98A, speeding up their movement during the synaptic contraction phase. Our findings reveal a new mechanism that regulates Rab4 vesicle transport and its impact on synapse stability. The study further suggests potential targets for controlling age-related dementia likely to be caused by excessive insulin signaling in neurons.

Like a photoreceptor cilium, the sensory cilia have a complex bipartite architecture containing 9+0 connecting cilium at the base and a singlet microtubule-supported, highly membranous outer segment, essential for the receptor display. How such diverse cilia morphology and underlying microtubule cytoskeleton develops remains unclear. Here we show that individual olfactory cilium, inside the large basiconic sensilla in developing Drosophila antenna, grows in episodic steps following several pulsatile influxes of tubulin. Each tubulin influx event is preceded by transient elevations of a microtubule-stabilising protein, the End-binding protein 1 (EB1). Additionally, EB1 is found to specifically interact with the tail domain of Drosophila KLP68D, an orthologue of the kinesin-2β motor subunit, in vitro . Finally, the loss of EB1 in olfactory neurons preceding the growth surges reduces the tubulin influx as well as arrests the olfactory cilia assembly and stability. These findings suggest a novel mechanism of bipartite cilia assembly.

Rab4 GTPase organizes endosomal sorting essential for maintaining the balance between recycling and degradative pathways. Rab4 localizes to many cargos whose transport in neurons is critical for regulating neurotransmission and neuronal health. Furthermore, elevated Rab4 levels in the CNS are associated with synaptic atrophy and neurodegeneration in Drosophila and humans, respectively. However, how the transport of Rab4-associated vesicles is regulated in neurons remains unknown. Using in vivo time-lapse imaging of Drosophila larvae, we show that activation of insulin signaling via Dilp2 and dInR increases the anterograde velocity, run length, and flux of Rab4 vesicles in the axons. Molecularly, we show that activation of neuronal insulin signaling further activates Vps34, elevates the levels of PI(3)P on Rab4-associated vesicles, recruits Klp98A (a PI(3)P-binding kinesin-3 motor) and activates their anterograde transport. Together, these observations delineate the role of insulin signaling in regulating axonal transport and synaptic homeostasis. Highlights: Dilp2-mediated insulin signaling activates anterograde transport of Rab4 vesicles Vps34 regulates anterograde velocity, run length and flux of Rab4 vesicles Local PI(3)P signaling on Rab4 vesicles regulates their motility in the axons PI(3)P production upon acute insulin stimulation recruits Klp98A on Rab4 vesicles

Toll-like and Interleukin-1/18 receptor resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD ⁺ hydrolysis and map the conformational effects of its interaction with NAD ⁺ using HDX-MS. NAD ⁺ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using 2pFLIM which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIR ^EA protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Cilium, a tiny microtubule‐based cellular appendage critical for cell signalling and physiology, displays a large variety of receptors. The composition and turnover of ciliary lipids and receptors determine cell behaviour. Due to the exclusion of ribosomal machinery and limited membrane area, a cilium needs adaptive logistics to actively reconstitute the lipid and receptor compositions during development and differentiation. How is this dynamicity generated? Here, we examine whether, along with the Intraflagellar‐Transport, targeted changes in sector‐wise lipid composition could control the receptor localisation and functions in the cilia. We discuss how an interplay between ciliary lipid composition, localised lipid modification, and receptor function could contribute to cilia growth and signalling. We argue that lipid modification at the cell‐cilium interface could generate an added thrust for a selective exchange of membrane lipids and the transmembrane and membrane‐associated proteins.

Kinesins, the microtubule-dependent mechanochemical enzymes, power a variety of intracellular movements. Regulation of Kinesin activity and Kinesin-Cargo interactions determine the direction, timing and flux of various intracellular transports. This review examines how phosphorylation of Kinesin subunits and adaptors influence the traffic driven by Kinesin-1, -2, and -3 family motors. Each family of Kinesins are phosphorylated by a partially overlapping set of serine/threonine kinases, and each event produces a unique outcome. For example, phosphorylation of the motor domain inhibits motility, and that of the stalk and tail domains induces cargo loading and unloading effects according to the residue and context. Also, the association of accessory subunits with cargo and adaptor proteins with the motor, respectively, is disrupted by phosphorylation. In some instances, phosphorylation by the same kinase on different Kinesins elicited opposite outcomes. We discuss how this diverse range of effects could manage the logistics of Kinesin-dependent, long-range intracellular transport.