Research Interests:
  • Mechanisms of membrane deformation
  • Endosomal membrane traffic and signaling
  • Endosomal membrane traffic and disease

Our lab is focused on understanding how neurons set up elaborate structures that are tailored to send and receive electrical signals over large distances and through complex networks of connections. Neurons undergo dynamic structural changes in response to external growth cues during development as well as learning and memory, and die back in the absence of positive growth cues. These growth cues are received at the cell surface and are trafficked into the cell via a network of membrane-bound compartments called endosomes.

We still do not understand the identity of the internal compartments from which growth cues signal, the special properties of those compartments that enable signaling to occur, and ultimately how the hundreds of proteins that make up the membrane traffic machinery can themselves be regulated to tune signaling up or down. These receptor trafficking events are implicated in neuronal diseases ranging from neurodegenerative disease to mental retardation and addiction, underlining the health importance of understanding how signal transduction is modulated by intracellular membrane traffic in neurons.

We use a combination of biochemistry, genetics, and live imaging in the fruit fly nervous system to unravel the molecular mechanisms by which the traffic of signaling receptors that control the architecture of synapses is regulated by interacting networks of membrane remodeling proteins. 

Mechanisms of membrane deformation

Figure 1. a) Liposome scalloping by the F-BAR protein Nwk [Becalska et al 2013]; magnified view below. b) Scalloping of a cell by Nwk. c) Model for membrane ridging by higher order Nwk F-BAR domain assemblies.

We are dissecting the mechanisms by which synaptic growth-regulating machinery that we have identified deforms membranes to drive traffic between endosomes. Nervous wreck (Nwk), a neuronal member of a family of F-BAR domain-containing membrane-deforming proteins, attenuates growth factor signaling from early endosomes. We recently discovered a unique and unexpected membrane deforming activity for Nwk, which forms membrane ridges that can be deformed by cytoskeletal forces (Becalska et al. 2013, Figure 2).

Figure 2. Diagram of Nwk activities and interactions

Nwk cooperates and interacts in vivo with many other membrane-associated proteins, including the membrane scission-driving GTPase dynamin (Figure 3). What remains lacking is an understanding of how these many membrane-remodeling factors co-assemble and work together to sculpt endosomes in vivo. We are using parallel biochemical approaches and high-resolution imaging in vivo to determine how complex networks of membrane remodeling proteins interact, cooperate and self-organize on organelles to control cargo traffic in neurons.

Endosomal membrane traffic and signaling

In Drosophila larvae, motor neurons innervate the body wall muscles in a stereotyped pattern, forming at their terminals an arbor of synaptic vesicle-containing boutons. Over the course of larval development, the muscle surface area grows 100-fold, requiring expansion of synaptic arbors to achieve muscle contraction. This growth is regulated by an integrated mechanism involving neuronal activity, retrograde signals, and anterograde signals. Mutations in proteins affecting the membrane traffic exhibit excessive bouton sprouting, which is thought to reflect a primary defect in the endosomal regulation of presynaptic signaling complexes involved in synaptic growth (Figure 3). We are studying how conserved proteins control this endocytosis, and finding points of regulation that are used to modulate membrane traffic and thus the output of synaptic growth pathways. 

Figure 3. Endocytic regulation of synaptic growth signaling

Left: Endocytic machinery (α-Nervous Wreck) surrounds sites of synaptic vesicle exocytosis (α-Brp) in larval boutons.
Middle: Endocytosis defects result in unconstrained growth of the larval NMJ.
Right: Activated Tkv receptor TkvQ199D) is relocalized to internal compartments by SNX163A (endocytosis mutant) relative to SNX16 (wild-type), resulting in increased signaling.

Figure 4. Confocal live imaging of a Drosophila larval motor axon showing movement of receptors.

We are devising time-resolved trafficking assays for imaging receptor-mediated endocytosis at the Drosophila NMJ, taking advantage of exciting new developments in live imaging by using spinning disk confocal microscopy (Figure 4). We have generated a panel of transgenic flies expressing fluorescently tagged markers of endocytosis so that we can image the path of receptors through the endosomal pathway after stimulation with labeled ligand. We can then measure the rate of receptor endocytosis in a wide variety of mutant synapses, to evaluate at what step synaptic growth is being perturbed. This system offers unprecedented experimental access to synaptic receptor-mediated endocytosis, since larval NMJs are stereotyped and large enough to image subcellular events by light microscopy in living animals over long periods of development.

Endosomal membrane traffic and disease

Intracellular membrane traffic serves to compartmentalize cellular signaling and biochemical events, and is both a prominent point of disease-related defects in neurodegenerative conditions including Alzheimer's disease (AD) and Amyotrophic Lateral Sclerosis (ALS), as well as a potential point of intervention. Healthy tissues maintain innervation by producing and releasing neurotrophic factors that control both the structure and function of the innervating neuron. These factors bind to neuronal receptors and drive their internalization into a "signaling endosome" that undergoes retrograde traffic to the cell body to convey pro-survival, differentiation, or pro-apoptotic signals to the nucleus. During neurodegenerative disease, neurons exhibit altered localization of neurotrophin receptors and may switch neurotrophin responsiveness from pro-survival to pro-apoptotic signaling. Neurotrophin signaling pathways may thus be effective points of intervention, particularly if methods can be found to modify these signaling pathways locally and specifically, for example at the level of intracellular traffic of their receptors. 

We are exploring the connection between disease phenotypes and these growth-signaling pathways, using genetics and live imaging to manipulate and visualize sub-cellular neuronal trafficking events in established Drosophila models of neurodegenerative disease. We are also probing the cellular functions of several conserved Drosophila homologs of genes that function in endosomal trafficking pathways and are mutated in human neurological disorders.