CRC 629 A15 - Regulation of the GTPase Rap1B during the differentiation of neurons

Basic data for this project

Description

The establishment of a polarized morphology and the functional specialization of different cellular compartments are essential steps in the differentiation of neurons. However, the initial signals that establish a cellular asymmetry and the pathways that subsequently translate this asymmetry into the development of multiple dendrites and a single axon are largely unknown. We use primary cultures of dissociated hippocampal neurons as a model system to elucidate the molecular mechanisms that establish the asymmetric organization of cellular structures. Shortly after plating, hippocampal neurons first extend lamellipodia (stage 1) and subsequently several processes, which are initially indistinguishable (stage 2) until one of them becomes the axon (stage 3). The molecular nature of the signal(s) that initiate cellular asymmetries and the negative feedback that blocks the formation of additional axons once an axon has been specified remain to be elucidated. We are investigating the role of the different GTPases in the establishment of neuronal polarity to understand the initial events that determine which neurite becomes the axon. We recently identified a small GTPase (Rap1B) that is specifically localized to the tip of a single neurite before it becomes distinguishable as an axon. Rap1B acts upstream of Cdc42 and the tripartite mPar3/mPar6/aPKC (Par) complex to determine which neurite will become the axon. It directs the recruitment of Cdc42 that is essential for the formation of axons. Expression of the constitutively active mutant Rap1BV12 induces the formation of multiple axons while its suppression by RNA interference results in the absence of axons. Our results show that the sequential activity of the GTPases Rap1B and Cdc42 is essential for the development of neuronal polarity. Initially, these GTPases are present in all neurites of unpolarized neurons but become restricted to a single neurite of late stage 2 neurons. Cdc42 coordinates the activity of different effectors and Rho-family GTPases to execute a program that results in the differentiation of neurons with molecularly distinct dendrites and axons. The establishment of polarity in hippocampal neurons. (a) The distribution of Rap1B (red), P-Akt (yellow), Cdc42 (green), Par3 (blue), and P-aPKC (magenta) during the differentiation of hippocampal neurons is shown schematically. Initially, the tips of all neurites are positive for Rap1B, P-Akt and Cdc42 (early stage 2). Rap1B is the first protein to become restricted to one neurite (late stage 2). P-Akt and Cdc42 are localized to the axon at stage 3. mPar3 first appears in all neurites at late stage 2 and becomes restricted to the axon in stage 3. P-aPKC is present only in the axon of stage 3 neurons. (b) The development of an axon in hippocampal neurons is initiated through intrinsic and/or extrinsic signals that lead to an enrichment of Rap1B in one of the neurites. Rap1B activity initiates the stabilization of Cdc42 in the prospective axon and its loss from the remaining neurites. Cdc42 cooperates with the Par complex to activate aPKC in the axon and probably also regulates the activity of Rho family GTPases that mediate axonal differentiation by polarizing the actin cytoskeleton and intracellular trafficking. We plan to address the following questions in the SFB 629: How is the GTPase Rap1B regulated by extrinsic and intrinsic factors? What is the molecular basis of the positive and negative feedback loops that mediate the establishment of neuronal polarity?