Role of Lamellipodin in fast, clathrin-independent endocytosis.

The human body is composed of billions of cells. In order for the body to function properly these cells have to communicate with each other. They communicate by sending out signals that can be recognized by "antenna", which are called "receptors", on the surface of other cells. When a signal reaches a receptor, the receptor becomes activated, which stimulates cells to react and change their behaviour. The signal is stopped by removal of the activated receptors from the cell surface by uptake into vesicles, small compartments that are surrounded by a membrane. This process is called "endocytosis". Endocytosis is important for the normal functioning of our body but if it "goes wrong" it can lead to disease. For example, in the case of wound healing, upon injury, cells in the wound send out signals to attract other cells to repair the wound. When these "repair" cells receive the signal, they actively migrate to the wound to close it. When the process of endocytosis is not working properly in the repair cells, wound healing may be delayed and this may contribute to many diseases such as diabetic foot ulcers, venous leg ulcers, and pressure ulceration (decubitus) of immobilised patients. Especially, in an ageing population pathological wound healing becomes a major challenge.
Cells send out signals by filling small vesicles inside the cell "X" with the compound that acts as a signal. The vesicle is then transported to the cell membrane of cell "X", the boundary of the cell with the outside world, and the content is released to the outside when the membrane of the vesicle fuses with the membrane of the cell "X". Nerve cells have many contacts with each other, called synapses, to relay signals to each other. This is the fundamental basis of how the brain works. At these synapses the vesicles with the stored signal is released to activate a neighbouring nerve cell "Y" by fusion of the vesicle with the membrane of the nerve cell "X". The signal is then diffusing to nerve cell "Y" where it activates receptors. Upon release of the signal the vesicle membrane has to be retrieved in order for the vesicle to be made again. The retrieval of the membrane is mediated again by endocytosis and is called synaptic vesicle recycling. Aberrant synaptic vesicle recycling impairs the communication of the nerve cells and this is thought to contribute to many neuropsychiatric diseases, which are major challenges to life long health and wellbeing such as epilepsy, depression, and schizophrenia.
We propose to study the basic mechanisms of endocytosis of receptors and synaptic vesicle recycling using single cells cultured in the laboratory. We will analyze how cells normally control the uptake of receptors using biochemical approaches. We will also visualize individual proteins recruited to the vesicles by labeling them with a fluorescent dye. This will allow us to analyze the recruitment in living cells in real time using a microscope. Our study might permit us to uncover the role of the different proteins for the uptake of the receptors thereby explaining the mechanism of this fundamental biological process. We will also be using a microscope to make movies of cells migrating in a dish while exposed to signals from other cells. We will use this assay to uncover how endocytosis may contribute to the directed movement of cells to the signal as it would normally happen during wound healing. The outcome of the project will give novel insights into the normal function of individual cells within our body and in the long run may provide the knowledge for the development of targeted therapies for diseases with altered endocytosis such as pathological wound healing as well as neuropsychiatric diseases.

Endocytosis is essential for homeostasis ensuring synaptic vesicle recycling and attenuation of epidermal growth factor receptor (EGFR) signalling, which controls directed migration during wound healing.
Lamellipodin (Lpd) binds endophilin to promote EGFR uptake via the actin effector Mena. It was recently reported that Lpd recruits endophilin to the leading edge of cells thereby inducing clathrin-independent, "Endophilin-positive assemblies (EPAs)" from which Fast, Endophilin-Mediated Endocytosis (FEME) occurs. FEME has also recently been implicated in synaptic vesicle recycling. However, FEMEs molecular mechanism remains enigmatic.
We found a novel interaction between Lpd and SNX9 and SNX9 localizes to EPAs. The BAR domains of endophilin and SNX9 bind to and induce different plasma membrane curvatures. We will test whether Lpd sequentially recruits SNX9 and endophilin to induce first shallow and later steep membrane invagination to facilitate FEME and investigate the mechanism of differential recruitment.
Antagonism between Arp2/3 activators and inhibitors might dynamically control actin polymerization during endocytosis. SNX9 binds the Arp2/3 complex and we will characterise its putative Arp2/3 inhibitory function. We will also test whether Lpd controls formation and endocytosis of EPAs to mediate EGFR uptake via SNX9, endophilin, the actin effectors Scar/WAVE, Arp2/3, N-WASP, or Mena and regulation of FEME.
Furthermore, we will analyse whether Lpd mediates fast, clathrin-independent synaptic vesicle recycling via endophilin and SNX9. We will also investigate whether FEME and subsequent EGFR recycling to the leading edge during 2D and 3D chemotaxis is mediated by Lpd, SNX9, endophilin, Scar/WAVE, and Mena, which may convert shallow gradients of growth factors to polarized, persistent behaviour driving chemotaxis.
Taken together, our experiments will reveal the molecular mechanism of FEME and how it mediates synaptic vesicle recycling and chemotaxis.

The work proposed here will uncover the molecular mechanism of a novel, clathrin-independent endocytosis pathway that has recently attracted much attention. Our work will also lead to an advance in our understanding of growth factor receptor endocytosis and synaptic vesicle recycling, biological processes that, when aberrantly regulated, contribute to pathological wound healing and neuropsychiatric disorders, respectively.

Given that the proposed work is discovery research, the primary immediate beneficiaries will be the wider academic bioscience community both in the UK and elsewhere, as detailed in the academic beneficiaries statement. The proposed work will contribute to worldwide academic advancement in this area. Furthermore, the researchers employed on the proposed grant would benefit by being trained in state-of-the-art skills which can be used both for academic and non-academic career paths (e.g. biotechnology and pharmaceutical industry).

Beneficiaries of the proposed work will also include those in the UK biotechnology and pharmaceutical industries interested in novel druggable targets to prevent pathological wound healing (such as decubitus and diabetic foot ulcers) and to treat neuropsychiatric disorders (such as epilepsy, depression, and schizophrenia).

The general public will also benefit from the proposed work thanks to public engagement activities aimed at both adults and (school) children participating in our established outreach scheme at King's College London http://www.kcl.ac.uk/ biohealth/study/outreach/index.aspx.