Research

Retrograde injury signaling in lesioned nerve

This line of research addresses a long-standing problem in the field of nerve regeneration: how does the cell body receive information about an injury from the distant lesion site in the axon.

This research line addresses a long-standing problem in the field of nerve regeneration: how does the cell body receive information about an injury from the distant lesion site in the axon. We started by examining involvement of nuclear import factors, showing that importin β1 mRNA is located in axons and translated locally upon injury.
The importins complex is transported retrogradely via an interaction with dynein, and blocking the process inhibits regeneration. Thus, regeneration is triggered by signals from the injury site that are transported by an importin/dynein complex (Hanz et al, 2003). We then used differential proteomics to search for signaling components of the complex, showing that soluble truncated forms of vimentin transport phosphorylated ERKs (pERK) in injured rat or mouse sciatic nerve, by linking pERK to the retrograde motor dynein via an association with importin β1.

Strikingly, vimentin protects the pERK from phosphatases en route, thus establishing a novel mechanism for long distance transport of an activated kinase (Perlson et al., 2005). In more recent work we demonstrated that Ran GTPase and its associated effectors regulate the formation of importin signaling complexes in injured axons, by providing a locally regulated ‘safety catch’ that prevents inappropriate importin association (Yudin et al., 2008). A comprehensive characterization of signaling to transcription networks after axonal injury revealed transcription factors and other regulators of the regeneration response that are trafficked by this mechanism (Michaelevski et al., 2010; Ben-Yaakov et al, 2012). Subcellular knockout of axonal importin β1 by specific targeting of a 3’UTR sequence confirmed the central role of local axonal synthesis of importins in retrograde injury signaling (Perry et al., 2012). These findings established novel roles for importins and their regulators in cytoplasmic signaling and transport, with broad implications for integration of cytoplasmic and nuclear transport mechanisms in both normal and injured cells.

In more recent work we have focused on how local translation is regulated in the axon. A number of RNA Binding Proteins (RBPs) traffic importin β1 and other key mRNAs to axons, most prominently an RBP called nucleolin (Perry et al., 2016). Nucleolin also transports mTOR mRNA to axons, and localized regulation of mTOR translation is critical for regulation of local protein synthesis after axon injury (Terenzio et al., 2018). Our current research priorities in this line are understanding the mechanisms that control subcellular localization of nucleloin, determining the roles of additional axonal RBPs, and elucidating additional regulatory mechanisms that control local translation in axons.

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Cell Length/Size Sensing

In recent years we expanded our focus from questions of how cells transport signals over intracellular distance to the question of how cells can actually sense or measure such distances, or in other words the fundamental cell biological issue of cell size homeostasis

How can cells can actually sense or measure intracellular distances, or can they use such mechanisms for cell size or length regulation?

We combined computational modeling and experimental approaches to propose a new mechanism for cell size/length sensing, based on motor driven frequency based signaling (Rishal et al., 2012). Two salient attributes of the mechanism are robustness arising from the frequency-based rather than quantity-based nature of the signal, and simplification of the spatial complexity of the problem due to directional restriction of signal propagation to the cytoskeleton.

The latter attribute essentially distils the three-dimensional problem of cell size sensing to a one-dimensional solution, bringing an intriguing new perspective to a fundamental problem in cell biology.

The top schematics depict the main features of the model, while the lower schematic shows an experimentally validated prediction. For further details please see Rishal et al., 2012.

More recently, we demonstrated that that motor-dependent mRNA localization regulates neuronal growth and cycling cell size (Perry et al., 2016). We found that the RNA-binding protein nucleolin is associated with importin β1 mRNA in axons. Perturbation of nucleolin association with kinesins reduces its levels in axons, with a concomitant reduction in axonal importin β1 mRNA and protein levels. Strikingly, subcellular sequestration of nucleolin or importin β1 enhances axonal growth and causes a subcellular shift in protein synthesis. Similar findings were obtained in fibroblasts. Thus, subcellular mRNA localization regulates size and growth in both neurons and cycling cells.

Our current efforts in this research line are focused on understanding how subcellular regulation of RNA localization enables cell size sensing and cell growth regulation. We are examining the mechanisms underlying subcellular localization of nucleolin, trying to identify nucleolin cargo RNAs required for size sensing, and taking different approaches to elucidate links between localized translation, size sensing and growth control.