In cell analysis of Myosin Va function in organelle transport.

Lead Research Organisation: University of Nottingham
Department Name: School of Life Sciences

Abstract

In order that the cells of our body function correctly they must establish and maintain a complex arrangement of specialised internal structures (organelles). The function of these organelles is essential for life e.g. energy production. Transport and communication between organelles is vital for both the normal function of organelles, cells and the whole body. In line with the importance of organelle transport blocks and other defects in these processes result in many common human diseases such as Cystic Fibrosis, hypercholesterolemia, neuro-degeneration and some forms of cancer. To allow organelle transport, cells have evolved a complex (protein-based) machinery that can move material between different organelles and areas of the cell. One class of proteins required for this are motor proteins that convert chemical energy, from food, into movement and transport cargo along subcellular protein ?tracks? known as the cytoskeleton. One of these motors, MyosinVa (MyoVa), is the focus of this proposal.
MyoVa is present in many cell types (man, animals, yeasts and plants) and transports a variety of cargo. Defects in its function have been implicated in the life-threatening human neurological and albinism diseases (Griscelli syndrome Type I and Elejalde syndrome). Thus understanding the mechanism of MyoVa function is critical to understanding the function of the human nervous and pigmentation systems. MyoVa has been studied extensively in artificial experimental environments outside the cell (in vitro) that have led to the discovery of many properties that make MyoVa an ideal cargo transporter. However, the cellular environment is much more complex than artificially created environments, so the importance of these motor properties in intracellular transport (in vivo) remains unclear.
In this study melanosome transport in melanocytes, pigment producing skin cells, will be used as a model system to determine which MyoVa properties are essential for cargo transport in living cells. Melanosomes are large, pigment containing organelles that are easily seen using a microscope. They are produced near the cell centre and are transported to the cell periphery in a process that requires MyoVa. This process is essential for skin pigmentation and protection from solar ultraviolet radiation. Loss of MyoVa causes defects in transport resulting in melanosomes clustering near the nucleus. This cellular defect results in skin and hair albinism.
The main experimental system used to address the importance of MyoVa properties in cells will be to introduce versions of MyoVa protein in which specific motor properties have been altered (mutated) into melanocytes lacking their own MyoVa. The altered (or mutant) motor proteins will then be tested for their ability to compensate for the loss of the native protein by restoring normal melanosome transport into the cell periphery. The results of this study will provide information relevant to improvements in human health and development of novel technology.

Technical Summary

Importance. Cytoskeleton tracks and motors are vital for intracellular organisation and transport by regulating organelle movement and distribution. In vitro data reveal that dimeric motor MyosinVa (MyoVa) ?walks? along actin filaments, indicating a cargo carrying function in cells. Meanwhile cellular studies indicate that it tethers cargo at the cortical actin. Also MyoVa interacts with other cytoskeleton components, suggesting that it may co-ordinate cytoskeleton structure and function.
Aims. The overall objective of this proposal is to determine the mechanism by which MyoVa functions in organelle transport in cells. Specifically it will determine to what extent MyoVa functions in cells as a motor or a tether (Aims1+2)? And how interactions with other cytoskeleton proteins and intra-molecular regulatory interactions affect its function (Aim3)?
Methods. These issues will be addressed using a recently established melanosome transport assay that reports MyoVa function in the movement of physiologically relevant and easily observable cargo (melanosomes) within primary mammalian cells (melanocytes). In this assay MyoVa, and mutants altered in structural and enzymatic properties e.g. dimerisation, are introduced into MyoVaKO melanocytes using adenovirus expression vectors. The functionality of introduced protein is then tested by assessing its ability to rescue loss of endogenous MyoVa, measured by observing redistribution of pigment from the perinuclear cluster into peripheral dendrites. Beyond this simple assay further functional tests will be performed to better understand the cellular role of the mutated part of the protein. For non-rescuing mutants the basis of loss of function will be further defined by testing cargo targeting and motor activity. For rescuing mutants more subtle alterations of function will be investigated by measuring the efficiency of rescue, indicated by the kinetics of rescue and the expression level required for rescue.
Applicant. The applicant has 10 years experience working with cultured melanocytes, and molecular biology techniques required to fully exploit this assay system. He has successfully developed similar complementation assays to investigate the function of Myosin activating proteins Mlph and Myrip. Additionally he maintains a network of collaborators who will provide essential reagents (e.g. libraries, cell lines, viral vectors and mutant mice).
Outcomes. This program will generate valuable information on the role of MyoVa and other motors by establishing their role as regulators of cytoskeleton activity, tethers or cargo carriers. Many motor proteins, including MyoVa, have been linked to human disease and advances in the basic understanding of their cellular function will aid the development of treatments of these diseases.

Publications

10 25 50
 
Title Myosin-Va deficient immortal melanocyte cell line. 
Description Myosin-Va plays essential non-redundant roles in organelle transport in the nervous system and pigment cells. In collaboration with Lluis Montoliu in CNB Madrid, Elena Sviderskaya St George's London and Miguel Seabra Imperial College London we have generated an immortal myosin-Va null melanocyte cell line from dilute20j myosin mutant mouse. 
Type Of Material Cell line 
Provided To Others? No  
Impact We have more clearly define the role of the actin and microtubule cytoskeleton transport systems in melanosome transport using these cells see http://www.ncbi.nlm.nih.gov/pubmed/25065759 for details. 
URL http://www.ncbi.nlm.nih.gov/pubmed/25065759
 
Description Generation of immortal mouse melanocyte cell lines 
Organisation Imperial College School of Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution We have co-ordinated the generation of immortal mouse skin melanocytes from mice. We then used the immortal cells to test the role of myosin-Va, actin, microtubules, Rab3GEP and fmn-1 in organelle transport.
Collaborator Contribution Our collaborator at Imperial College London supplied the myosin-Va and Rab3GEP deficient mice. our collaborator at Centro Nacional de Biotecnologia (CNB-CSIC) crossed these myosin-Va KO mice with p16INK4A null mice and selected for double mutant progeny and intercrossed these to obtain double homozygous mutant pups. Our collaborator at St. George's, University of London then derive immortal cultures of melanocytes from the skins of these mice. Our collaborator at Institute Pasteur has provided fmn1 mutant mice.
Impact The main outcome of this collaboration is publication. http://www.ncbi.nlm.nih.gov/pubmed/25065759 https://www.ncbi.nlm.nih.gov/pubmed/28490438 In addition these melanocytes are available for use by other researchers from the Wellcome Trust functional genomics cell bank at St George's University of London http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm
Start Year 2012
 
Description Generation of immortal mouse melanocyte cell lines 
Organisation Pasteur Institute, Paris
Country France 
Sector Academic/University 
PI Contribution We have co-ordinated the generation of immortal mouse skin melanocytes from mice. We then used the immortal cells to test the role of myosin-Va, actin, microtubules, Rab3GEP and fmn-1 in organelle transport.
Collaborator Contribution Our collaborator at Imperial College London supplied the myosin-Va and Rab3GEP deficient mice. our collaborator at Centro Nacional de Biotecnologia (CNB-CSIC) crossed these myosin-Va KO mice with p16INK4A null mice and selected for double mutant progeny and intercrossed these to obtain double homozygous mutant pups. Our collaborator at St. George's, University of London then derive immortal cultures of melanocytes from the skins of these mice. Our collaborator at Institute Pasteur has provided fmn1 mutant mice.
Impact The main outcome of this collaboration is publication. http://www.ncbi.nlm.nih.gov/pubmed/25065759 https://www.ncbi.nlm.nih.gov/pubmed/28490438 In addition these melanocytes are available for use by other researchers from the Wellcome Trust functional genomics cell bank at St George's University of London http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm
Start Year 2012
 
Description Generation of immortal mouse melanocyte cell lines 
Organisation Spanish National Centre for Biotechnology
Country Spain 
Sector Public 
PI Contribution We have co-ordinated the generation of immortal mouse skin melanocytes from mice. We then used the immortal cells to test the role of myosin-Va, actin, microtubules, Rab3GEP and fmn-1 in organelle transport.
Collaborator Contribution Our collaborator at Imperial College London supplied the myosin-Va and Rab3GEP deficient mice. our collaborator at Centro Nacional de Biotecnologia (CNB-CSIC) crossed these myosin-Va KO mice with p16INK4A null mice and selected for double mutant progeny and intercrossed these to obtain double homozygous mutant pups. Our collaborator at St. George's, University of London then derive immortal cultures of melanocytes from the skins of these mice. Our collaborator at Institute Pasteur has provided fmn1 mutant mice.
Impact The main outcome of this collaboration is publication. http://www.ncbi.nlm.nih.gov/pubmed/25065759 https://www.ncbi.nlm.nih.gov/pubmed/28490438 In addition these melanocytes are available for use by other researchers from the Wellcome Trust functional genomics cell bank at St George's University of London http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm
Start Year 2012
 
Description Generation of immortal mouse melanocyte cell lines 
Organisation St George's University of London
Country United Kingdom 
Sector Academic/University 
PI Contribution We have co-ordinated the generation of immortal mouse skin melanocytes from mice. We then used the immortal cells to test the role of myosin-Va, actin, microtubules, Rab3GEP and fmn-1 in organelle transport.
Collaborator Contribution Our collaborator at Imperial College London supplied the myosin-Va and Rab3GEP deficient mice. our collaborator at Centro Nacional de Biotecnologia (CNB-CSIC) crossed these myosin-Va KO mice with p16INK4A null mice and selected for double mutant progeny and intercrossed these to obtain double homozygous mutant pups. Our collaborator at St. George's, University of London then derive immortal cultures of melanocytes from the skins of these mice. Our collaborator at Institute Pasteur has provided fmn1 mutant mice.
Impact The main outcome of this collaboration is publication. http://www.ncbi.nlm.nih.gov/pubmed/25065759 https://www.ncbi.nlm.nih.gov/pubmed/28490438 In addition these melanocytes are available for use by other researchers from the Wellcome Trust functional genomics cell bank at St George's University of London http://www.sgul.ac.uk/depts/anatomy/pages/WTFGCB.htm
Start Year 2012
 
Description Investigation of myosin-Va and spire actin assembly proteins in organelle transport 
Organisation University of Regensburg
Department Department of Neurology
Country Germany 
Sector Academic/University 
PI Contribution We have test the function of experimental spire type actin assembly proteins in an organelle transport assay in cells.
Collaborator Contribution Our collaborator has supplied us with vectors allowing expression of mutant/experimental spire proteins in mammalian cells which we are testing in our cellular assays.
Impact See the above paper investigating the co-ordination of myosin-V and spire proteins via the small GTPase Rab11.
Start Year 2015
 
Description Investigation of the mechanism of myosin-Va activation by cargo adaptor protein melanophilin. 
Organisation Technical University of Munich
Department Department of Physics
Country Germany 
Sector Academic/University 
PI Contribution Our collaborators are interested in understanding how cargo associated adaptor proteins e.g. melanophilin, regulate the activity of motor proteins e.g. myosin-Va in vitro. We have carried out complementary experiment in cells.
Collaborator Contribution They have generated purified motor proteins and adaptors and purified organelles from cells. They have use light microscopy to analyse the movements of motors and cargo in vitro. They have also assisted us by examining the ability of cargo to assemble actin tracks directly at the membrane using microscope based assays and purified components.
Impact Manuscript submitted to PNAS
Start Year 2015
 
Description Myristoyl transferase inhibitors and actin cytoskeleton 
Organisation Imperial College London
Department Department of Chemistry
Country United Kingdom 
Sector Academic/University 
PI Contribution We have identified a candidate substrate for NMT whose activity is required for the integrity of the actin cytoskeleton and melanosome transport in melanocytes.
Collaborator Contribution They have supplied us with highly specific inhibitors of NMT and an analogue labelling strategy that will allow us to confirm directly whether the candidate is indeed an NMT substrate.
Impact A manuscript describing the results of our studies is being prepared for submission to a peer reviewed journal.
Start Year 2017
 
Description Understanding the structure of the actin cytoskeleton in melanocytes using electron microscopy 
Organisation University College London
Department Institute of Ophthalmology UCL
Country United Kingdom 
Sector Academic/University 
PI Contribution We provide cultures of melanocytes to a collaborator at UCL who is using electron microscopy to analyse the structure and distribution of F-actin associated with pigmented melanosomes. The aim of the project is to understand how short 1micrometer actin filaments drive transport of these organelles over long distances 10-20 micrometers in melanocytes.
Collaborator Contribution The collaborator is using scanning electron microscopy to investigate the structure of actin in melanocytes provided by us and also in situ labelling using fluorescent actin to understand the distribution of F-actin assembly sites in these cells.
Impact A manuscript describing our results is in preparation.
Start Year 2017
 
Description Investigating the role of cytoskeleton motors in organelle transport 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact These sessions with A-level students stimulated discussion of the molecular mechanism underlying biological phenomenon specifically related to the role played by motor proteins and the cytoskeleton in organelle transport.

These sessions were conducted as part of the Universities widening participation program and Soton trust summerschools. Students attending these sessions have then applied to the University to study Life Science.
Year(s) Of Engagement Activity 2012,2013,2014
 
Description University of Nottingham Mayfest 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact University open day allowed members of the public to use a fluorescence microscopy to look at DNA in cancer cells. Also cytoskeleton proteins.

I engaged in interesting discussions with member of the public about basic science and molecular mechanism of disease including cancer.
Year(s) Of Engagement Activity 2012,2013,2014