High resolution imaging combined with A pH-sensitive probe to monitor exocytosis in growth cones.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The development of mammalian nervous system is a multistep process.

After neuronal differentiation, the newly differentiated neurons have to extend their axons along long distances in order to find their final destination. Along their journey, several guidance molecules guide their way, giving them instructions on the right way to go to form a correct brain circuitry. This process of guiding axons to the right destination is called axon guidance.

Growth cones are at the tip of a growing axon. They explore the environment, “feel” through their receptors. The binding of guidance molecules to their receptor triggers a signaling inside the cell -a chain of events involving several proteins- and a neuron behaves accordingly to the instructions received.

Several different molecules are involved in axon guidance, either attracting or repulsing the axons. Amongst these, netrin is an attractive molecule that by binding to its receptor DCC, activates a signaling cascade that changes the shape of the growth cone, affects the membrane dynamics, and attract the developing axon.

It has been proposed that attraction of a neuron to a guidance molecule is linked to exocytosis. Exocytosis is the fusion of a vesicle -a structure made of a membrane enveloping some cellular material- with the plasma membrane to release cellular components in the extracellular space.

How can we see what happens exactly at the membrane itself after netrin/DCC binding?

The tools. In order to see what happens at the membrane of a growth cone during guidance, the authors use a fluorescent probe to monitor little vesicles.

Synapto-pHluorin is a fluorescent probe consisting of a GFP (green fluorescent protein) modified in order to make it sensitive to pH. Inside the vesicle the pH is very acidic and that prevents the fluorescence of the probe. However, when the vesicle fuses with the plasma membrane, it exposes its content to the extracellular space, which has a different pH (more neutral). Now, at neutral pH the probe becomes fluorescent: many fluorescent spots appear on the surface of the growth cone.

Exocytosis in developing growth cones. After expressing this probe in hippocampal neurons in vitro, they imaged growth cones over time, to check carefully how and where exocytosis -fusion of the vesicles with the plasma membrane- happened. Thanks to Synapto-pHluorin, every time a vesicle fused with the plasma membrane a fluorescent spot appeared on the growth cone. Sometimes clusters of several vesicles fused together, resulting into large fluorescent spots appearing , sometimes only few single vesicles were fused with the plasma membrane, resulting into smaller fluorescence spots.

The growth cone has a particular structure with small protrusions called filopodia, which are highly dynamic, continuously moving and changing size, exploring the environment.  The change in size or shape of growth cones or filopodia often corresponded with vesicles fusion with the membrane.

These spontaneous events of exocytosis happened throughout the growth cones, but mainly at the periphery and in filopodia. Rather than random, they followed a certain pattern: moments of highly frequent exocytosis events followed by a low dynamic moment.

Exocytosis induced by netrin. Exocytosis happened more frequently if the cells were treated with the guidance molecule netrin.

What happens inside the cell stimulated by netrin to increase the frequency of exocytosis at the growth cone?

Netrin/DCC signaling inside the cells is linked to SFK and ERK1\2 proteins and their downstream pathways. Indeed, netrin stimulation causes the increased phosphorylation level of these two proteins, and when these two proteins are blocked with specific chemical inhibitors, netrin stimulation does not have any effect on the dynamics of vesicles, showing that netirn-induced exocytosis requires the activity of SFK and ERK1\2.

Exocytosis is linked to intracellular calcium concentration. During axon elongation, calcium concentration increases and decreases periodically. In particular, netrin stimulation has been associated with these temporary changes of calcium concentration that are called calcium transients. By imaging at the same time exocytosis and calcium concentration in growth cones, they showed that upon netrin stimulation these events of vesicle fusion with plasma membrane are parallel to a local increase of frequency of calcium transients in some parts of the growth cones, suggesting that calcium may modulate exocytosis in netrin-stimulated growth cones.

Conclusion.

A high resolution and thorough analysis of vesicle fusion with the plasma membrane in neurons in culture with fluorescent probe, showed that netrin binding to DCC triggers exocytosis, through the activation of SFK and ERK1/1 pathway, probably due to an increased frequency of calcium transients, suggesting that exocytosis may be the mechanism controlling axon extension upon attractive guidance molecules.

     

Reference: Regulation of patterned dynamics of local exocytosis in growth cones by netrin-1. Ros O, Cotrufo T, Martínez-Mármol R, Soriano E. The journal of neuro science.