The glycine arginine-rich domain of the RNA-binding protein nucleolin regulates its subcellular localization.

Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein-protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid-liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA-RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease.

Spatial-specific functions in retrograde neuronal signalling.

Neurons are highly polarized cells, possessing long axons that can extend to more than 1-m long in adult humans. In order to survive and maintain proper functions, neurons have to respond accurately in both space and time to intracellular or intercellular cues. The regulation of these comprehensive responses involves ligand-receptor interactions, trafficking and local protein synthesis. Alterations in these mechanisms can lead to cellular dysfunction and disease. Although studies on the transport and localization of signalling endosomes along the axon have shed light on some central pathways of neuronal survival and growth as well as synapse function, little is known about the spatiotemporal mechanisms that allow the same molecule to signal differently at diverse subcellular locations and in specific neuronal populations. In this review, we will provide an overview of retrograde axonal signalling mechanisms and discuss new advances in our understanding of the spatial-specific regulation of neuronal signalling and functions, mechanisms that allow the same signal to have a different effect in another subcellular location.

A compartmentalized microfluidic neuromuscular co-culture system reveals spatial aspects of GDNF functions.

Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF--facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

Rabies Virus Hijacks and accelerates the p75NTR retrograde axonal transport machinery.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.

EspM inhibits pedestal formation by enterohaemorrhagic Escherichia coli and enteropathogenic E. coli and disrupts the architecture of a polarized epithelial monolayer.

Enterohaemorrhagic Escherichia coli and enteropathogenic E. coli are enteropathogens characterized by their ability to induce the host cell to form actin-rich structures, termed pedestals. A type III secretion system, through which the pathogens deliver effector proteins into infected host cells, is essential for their virulence and pedestal formation. Enterohaemorrhagic E. coli encodes two similar effectors, EspM1 and EspM2, which activate the RhoA signalling pathway and induce the formation of stress fibres upon infection of host cells. We confirm these observations and in addition show that EspM inhibits the formation of actin pedestals. Moreover, we show that translocation of EspM into polarized epithelial cells induces dramatic changes in the tight junction localization and in the morphology and architecture of infected polarized monolayers. These changes are manifested by altered localization of the tight junctions and 'bulging out' morphology of the cells. Surprisingly, despite the dramatic changes in their architecture, the cells remain alive and the epithelial monolayer maintains a normal barrier function. Taken together, our results show that the EspM effectors inhibit pedestal formation and induce tight junction mislocalization as well as dramatic changes in the architecture of the polarized monolayer.

Multiple layers of spatial regulation coordinate axonal cargo transport.

Nerve axons are shaped similar to long electric wires to quickly transmit information from one end of the body to the other. To remain healthy and functional, axons depend on a wide range of cellular cargos to be transported from the neuronal cell body to its distal processes. Because of the extended distance, a sophisticated and well-organized trafficking network is required to move cargos up and down the axon. Besides motor proteins driving cargo transport, recent data revealed that subcellular membrane specializations, including the axon initial segment at the beginning of the axon and the membrane-associated periodic skeleton, which extends throughout the axonal length, are important spatial regulators of cargo traffic. In addition, tubulin modifications and microtubule-associated proteins present along the axonal cytoskeleton have been proposed to bias cargo movements. Here, we discuss the recent advances in understanding these multiple layers of regulatory mechanisms controlling axonal transport.

Combined kinesin-1 and kinesin-3 activity drives axonal trafficking of TrkB receptors in Rab6 carriers.

Neurons depend on proper localization of neurotrophic receptors in their distal processes for their function. The Trk family of neurotrophin receptors controls neuronal survival, differentiation, and remodeling and are well known to function as retrograde signal carriers transported from the distal axon toward the cell body. However, the mechanism driving anterograde trafficking of Trk receptors into the axon is not well established. We used microfluidic compartmental devices and inducible secretion assay to systematically investigate the retrograde and anterograde trafficking routes of TrkB receptor along the axon in rat hippocampal neurons. We show that newly synthesized TrkB receptors traffic through the secretory pathway and are directly delivered into axon. We found that these TrkB carriers associate and are regulated by Rab6. Furthermore, the combined activity of kinesin-1 and kinesin-3 is needed for the formation of axon-bound TrkB secretory carriers and their effective entry and processive anterograde transport beyond the proximal axon.

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers.

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER-ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins' role in ER-to-Golgi transport.

Wnt Signaling Directs Neuronal Polarity and Axonal Growth.

The establishment of neuronal polarity is driven by cytoskeletal remodeling that stabilizes and promotes the growth of a single axon from one of the multiple neurites. The importance of the local microtubule stabilization in this process has been revealed however, the external signals initiating the cytoskeletal rearrangements are not completely understood. In this study, we show that local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth. We found that in the early stages of neuronal polarization, Wnt3a accumulates in one of the neurites of unpolarized cells and thereby could determine axon positioning. Subsequently, Wnt3a localizes to the growing axon, where it activates the canonical Wnt pathway and controls axon positioning and axonal length. We propose a model in which Wnt3a regulates the formation and growth of the axon by activating local intracellular signaling events leading to microtubule remodeling.

The receptor tyrosine kinase TrkB signals without dimerization at the plasma membrane.

Tropomyosin-related tyrosine kinase B (TrkB) is the receptor for brain-derived neurotrophic factor (BDNF) and provides critical signaling that supports the development and function of the mammalian nervous system. Like other receptor tyrosine kinases (RTKs), TrkB is thought to signal as a dimer. Using cell imaging and biochemical assays, we found that TrkB acted as a monomeric receptor at the plasma membrane regardless of its binding to BDNF and initial activation. Dimerization occurred only after the internalization and accumulation of TrkB monomers within BDNF-containing endosomes. We further showed that dynamin-mediated endocytosis of TrkB-BDNF was required for the effective activation of the kinase AKT but not of the kinase ERK1/2. Thus, we report a previously uncharacterized mode of monomeric signaling for an RTK and a specific role for the endosome in TrkB homodimerization.

Compartmental microfluidic system for studying muscle-neuron communication and neuromuscular junction maintenance.

Molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. Disruption in the structure and function of NMJs is a hallmark of various neurodegenerative processes during both development and pathological events. Still due to the complexity of this process, it is very difficult to elucidate the cellular mechanisms underlying it, generating a keen interest for developing better tools for investigating it. Here we describe a simplified method to study mechanisms of NMJs formation, maintenance and disruption. A spinal cord explant from mice expressing the Hb9::GFP motoneuron marker is plated on one side of a compartmental chamber, and myotubes derived from muscle satellite progenitor cells are plated on the other. The GFP labeled motoneurons extend their axons via microgrooves in the chamber to innervate the muscle cells and to form functional in-vitro NMJs. Next we provide procedures to measure axon growth and to reliably quantify NMJ activity using imaging of both muscle contractions and fast intracellular calcium changes. This platform allows precise control, monitoring and manipulation of subcellular microenvironments. Specifically, it enables to distinguish local from retrograde signaling mechanisms and allows restricted experimental intervention in local compartments along the muscle-neuron route.

Developmental Axon Pruning Requires Destabilization of Cell Adhesion by JNK Signaling.

Developmental axon pruning is essential for normal brain wiring in vertebrates and invertebrates. How axon pruning occurs in vivo is not well understood. In a mosaic loss-of-function screen, we found that Bsk, the Drosophila JNK, is required for axon pruning of mushroom body γ neurons, but not their dendrites. By combining in vivo genetics, biochemistry, and high-resolution microscopy, we demonstrate that the mechanism by which Bsk is required for pruning is through reducing the membrane levels of the adhesion molecule Fasciclin II (FasII), the NCAM ortholog. Conversely, overexpression of FasII is sufficient to inhibit axon pruning. Finally, we show that overexpressing other cell adhesion molecules, together with weak attenuation of JNK signaling, strongly inhibits pruning. Taken together, we have uncovered a novel and unexpected interaction between the JNK pathway and cell adhesion and found that destabilization of cell adhesion is necessary for efficient pruning.

Real-time sensing of enteropathogenic E. coli-induced effects on epithelial host cell height, cell-substrate interactions, and endocytic processes by infrared surface plasmon spectroscopy.

Enteropathogenic Escherichia coli (EPEC) is an important, generally non-invasive, bacterial pathogen that causes diarrhea in humans. The microbe infects mainly the enterocytes of the small intestine. Here we have applied our newly developed infrared surface plasmon resonance (IR-SPR) spectroscopy approach to study how EPEC infection affects epithelial host cells. The IR-SPR experiments showed that EPEC infection results in a robust reduction in the refractive index of the infected cells. Assisted by confocal and total internal reflection microscopy, we discovered that the microbe dilates the intercellular gaps and induces the appearance of fluid-phase-filled pinocytic vesicles in the lower basolateral regions of the host epithelial cells. Partial cell detachment from the underlying substratum was also observed. Finally, the waveguide mode observed by our IR-SPR analyses showed that EPEC infection decreases the host cell's height to some extent. Together, these observations reveal novel impacts of the pathogen on the host cell architecture and endocytic functions. We suggest that these changes may induce the infiltration of a watery environment into the host cell, and potentially lead to failure of the epithelium barrier functions. Our findings also indicate the great potential of the label-free IR-SPR approach to study the dynamics of host-pathogen interactions with high spatiotemporal sensitivity.