Role of kinesin light chain 1 in binding to specific cargoes.

Lead Research Organisation: University of Manchester
Department Name: School of Biological Sciences

Abstract

Cells of all organisms except bacteria contain filaments, called microtubules, that act as tracks for the transport of material from one region to another. This vital traffic is carried by proteins that 'walk' along microtubules, acting as minute motors that move many different cargoes. There are two families of microtubule motor proteins, the kinesins and dyneins, with most kinesins carrying cargo away from the cell centre towards the cell periphery. These motors and the cargoes they carry are absolutely vital for the health of nerve cells and for brain function, since mutations in them can cause or contribute to diseases such as muscular dystrophy, Alzheimer's disease, Huntington's disease, spastic paraplegia and schizophrenia. Their function is vital in all cells of the body, not just neurons.

Kinesin-1 transports many different kinds of cargo, ranging from membrane organelles through to cytoskeletal proteins. It is made up of two types of protein subunits: the KIF5 subunits provide the motor activity, while the kinesin light chains (KLCs) bind to cargo proteins and also control kinesin's activity. The KLCs come in several different types, so one possibility is that each cargo uses kinesin-1 containing a particular KLC. There are four KLC genes, with the KLC1 gene being alternatively spliced to generate at least 17 different proteins (isoforms) that vary in amino acid sequence only at one end, their C-terminal. Our functional studies showed that two isoforms, KLC1B and KLC1D, each control the movement of different membrane types, supporting the idea that KLC variation is crucial for cargo selection. KLC1 splicing is important physiologically, as changes in the levels of certain splice forms have been linked to Alzheimer's disease and schizophrenia. Altogether, it is clear that KLCs are central to kinesin-1 cargo binding and subsequent activation. However, we do not fully understand the importance of different KLCs-and particularly KLC1 isoforms-in kinesin-1 function. As a first step, we must identify the cellular cargoes to which they bind, and the specific proteins that recruit them to those cargoes.

To begin to do this, we have used a technique called BioID that adds a biotin molecule to any protein in the near neighbourhood of a specific KLC isoform. Biotinylated proteins are then isolated and identified using mass spectrometry. We found that KLC1D, but not KLC2 or 3, was in close proximity to three proteins involved in endocytosis. This pathway is the route by which cells take up material (nutrients and growth factors, for example) from outside the cell. Two of the KLC1D near-neighbours, SNX1 and CCDC22, are involved in sorting material that should be recycled from that to be degraded. The third, BIRC6, is needed for cells to divide into two after cell division in a process called cytokinesis. A major goal of this project is to test how kinesin-1 containing the KLC1D isoform contributes to protein sorting at the early endosome, focussing on the two pathways that require SNX1 and CCDC22. We will also investigate kinesin's role in cytokinesis. To do this, we will make a new kinesin tool-box that will allow us to remove KLC1 rapidly from cells, or replace the cell's kinesin on cargo with an inactive version that lacks the motor domains. We will use light microscopy and biochemical assays to monitor what happens to endocytosis after disrupting kinesin function. We will also determine if SNX1, CCDC22 and BIRC6 can bind directly to KLC1D, and if so, dissect the regions of each protein needed for this binding. Finally, we will use BioID to test if KLC1 isoform do indeed target kinesin-1 to specific organelles, using three additional KLC1 splice forms. Overall, this project will provide important insight into how KLC1 splicing affects kinesin function and association with specific cargoes.

Technical Summary

Kinesin-1-driven transport is essential for cell function in most eukaryotes, due in part to the wide variety of cargoes it carries, ranging from membrane organelles through to cytoskeletal proteins. Kinesin-1 consists of two motor subunits and two light chains (KLCs). Multiple genes encode each subunit, with further complexity provided by alternative splicing of the KLC1 gene, giving isoforms that differ widely in their C-terminal domain and have been implicated in human disease. An attractive hypothesis is that this variation enables kinesin-1 to bind to and transport such a plethora of cargo. Our overall aim is to uncover the specific roles palyed by KLC1 isoforms in membrane traffic, focusing on the early endocytic pathway.

Using a mass spectrometry-based proximity biotinylation approach (BioID), we identified three endosome-associated proteins as near-neighbours of KLC1D, but not KLC2 or 3. Two of these play key roles in sorting and recycling material from the endosome via the ESCPE-1 and Retriever pathways while the third is involved in the recruitment of recycling endosomes to the midbody as an essential step in cytokinesis. We will determine how kinesin containing KLC1D contributes to these vital processes. To do this, we will establish a novel tool-box for manipulating the function of KLC1, including a new dominant-negative construct approach, and the development of a cell line with a degron tag inserted into the KLC1 gene. Upon auxin addition, the KLC1 protein will be rapidly degraded, giving an acute means of depleting kinesin containing KLC1. These tools will be very useful right across the kinesin field. We will also test if other KLC1 isoforms have shared or distinct roles compared to KLC1D by extending our BioID approach to identify specific membrane cargoes and interactors of three additional key KLC1 splice forms. Altogether, this project will provide the first molecular insight into how KLC1 splicing affects kinesin function.

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