The role of microtubule plus-end capture at cortical sites in the assembly of apico-basal microtubules in polarised epithelial cells.
Lead Research Organisation:
University of East Anglia
Department Name: Biological Sciences
Abstract
Microtubules are tubular structures, which are important for many cellular functions including the transport of vesicles and molecules within cells. It is therefore very important that the microtubules are assembled and positioned correctly within cells. The specific pattern that microtubules form within cells vary depending on cell type and function. Most animal cells have a radial array of microtubules anchored at a centrally located structure called the centrosome. In these cells the centrosome's job is to start off the assembly of the microtubules and to keep them tightly bound to it. However, many specialised cells like polarised epithelial cells found in the gut and kidneys arrange their microtubules in a different way. Here most of the microtubules are anchored at the cell apex away from the centrosome and run towards the base forming a so called apico-basal array. Our recent findings suggest that the apico-basal microtubules are assembled at the centrosome, released and moved to apical sites where they are captured and anchored. The aim of this project is to find out whether the microtubules first grow out from the centrosome, make contact with the cell cortex, are released from the centrosome and then move downward by the pulling action of dynein motor proteins located at the cortex. The end result would be that one end (plus-end) of the microtubules is pulled down to the cell base while the other end (minus-end) becomes anchored at the cell apex. We want to find out whether proteins such as EB1 and CLIP-170, which stick to the growing end of microtubules, are important for the capture by dynein or other proteins at the cortex. It is very important to know if for example CLIP-170 is vital for the normal assembly of the microtubule in real life. We will therefore analyse tissue from the inner ear (cochlea) isolated from mice, which do not produce CLIP-170. Finally, we would also like to know whether thin filaments known as actin help the microtubules to get to their destination. We will use special fluorescent (glowing) molecules called GFPs linked to proteins so that the microtubules or their plus-ends glow and we will make movies of their movements over time and look at the localisation of these important proteins using fluorescent dyes and specialised microscopes. Microtubules are clearly vital for the normal function of a cell and we need to establish how they are organised and controlled before we can unravel the causes and consequences of many diseases.
Technical Summary
This project will capitalise on our recent very promising findings concerning the assembly and organisation of non-centrosomal Mts in epithelial cells. It will benefit from the use of a combination of inner ear tissue and cell culture model systems and an exciting opportunity to analyse the role of mammalian CLIPs and CLASPs in vivo. Epithelial polarisation involves major reorganisation of the microtubule cytoskeleton from a radial centrosomally focused array to an apico-basal array associated with apical non-centrosomal anchoring sites. Correct assembly and positioning of these non-centrosomal microtubules is vital for many of the highly specialised functions performed by differentiated cells. Evidence from the applicant's lab. suggests that the non-centrosomal microtubules originate from the centrosome and that a microtubule 'release and capture' mechanism is exploited in many epithelial cells as a means of generating these microtubules. This proposal specifically sets out to determine whether microtubule plus-end capture at cortical sites is a vital intermediate step in the assembly of apico-basal arrays in polarised epithelial cells. The aims are to determine whether dynein acts as a protein linking microtubule +TIPs such as EB1 and CLIP-170 to the cell cortex and act as a motor protein pulling released microtubules towards the periphery and cell base. The aim is also to resolve whether microtubule-actin filaments interactions are important for microtubule guidance towards the cortical sites. Furthermore, we aim to identify protein interacting partners for EB1 and CLIP-170 in polarised epithelial cell. This investigation will use GFP- and RNAi-technology, protein biochemistry and high resolution widefield fluorescence, confocal and electron microscopy to pursue these objectives. Findings from this proposal will not only help to identify the molecular mechanisms involved in the assembly of non-centrosomal Mt arrays but also advance our understanding of the role of Mt plus-end cortical interactions in cells generally.
People |
ORCID iD |
Mette Mogensen (Principal Investigator) |
Publications
Bellett G
(2009)
Microtubule plus-end and minus-end capture at adherens junctions is involved in the assembly of apico-basal arrays in polarised epithelial cells.
in Cell motility and the cytoskeleton
Dahm R
(2007)
Reorganization of centrosomal marker proteins coincides with epithelial cell differentiation in the vertebrate lens.
in Experimental eye research
Goldspink DA
(2013)
The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation.
in Journal of cell science
Kohlmaier G
(2009)
Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP.
in Current biology : CB
Moss DK
(2007)
Ninein is released from the centrosome and moves bi-directionally along microtubules.
in Journal of cell science
Wagstaff LJ
(2008)
Multicellular rosette formation during cell ingression in the avian primitive streak.
in Developmental dynamics : an official publication of the American Association of Anatomists
Description | The overall aim of the project was to investigate the role of microtubule plus-end cortical interactions in microtubule organisation and particularly in the assembly of non-centrosomal apico-basal arrays in polarised epithelial cells. We established that the microtubule array that runs from the apex to the base in elongated epithelial cells originate from the centrosome (the main microtubule organising centre - MTOC) but are subsequently relocated and anchored at so called non-centrosomal organising centre (n-MTOC). Furthermore, we established that an extended radial array initially forms prior to the assembly of the apico-basal array and that microtubule plus-end cortical interaction is critical for this process. The microtubule associated proteins EB1, EB3 and CLIP-170 were shown to target cell to cell contacts known as adherens junctions and electron microscopy showed ends of microtubules associating with these junctions. Depletion data revealed that CLIP-170 is important in the establishment of apico-basal microtubule arrays in polarised epithelial cells and that this is likely to involve microtubule cortical capture. We have subsequently ( in a follow on project using knockout mice ) found that CLIP170 is not essential for apico-basal polarisation suggesting that a compensation mechanism exist which is not evident in 2D cell culture systems. A release and capture model involving both microtubule plus-end and minus-end capture at adherens junctions was proposed for the assembly of non-centrosomal apico-basal microtubule arrays. |
Exploitation Route | Some of the findings from this project were taken forward in a subsequent grant |
Sectors | Agriculture, Food and Drink,Chemicals,Communities and Social Services/Policy,Digital/Communication/Information Technologies (including Software),Education,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology |
Description | The findings from this research have been presented at conferences, seminars as well as to the public. A significant part has been published in high impact journals. Some of our findings have led to further applications to the BBSRC and other organisations of which some have been awarded including PhD studentships and project grants. The postdoc received extensive training in technical aspects as well as presentation, teaching and communication skill. She attended several national and international conferences where she presented her work. She was a co-applicant on a grant application for a PhD studentship to the BigC Appeal (cancer charity) which was awarded and she co-supervised the student. She also engaged in outreach programs demonstrating widefield fluorescence microscopy to six form students and helped on visit days for prospective students. She obtained a permanent research position in industry concerned with development of biological therapies to promote healing and pain relief six months before the end of the project. |
First Year Of Impact | 2008 |
Sector | Digital/Communication/Information Technologies (including Software),Education,Other |
Impact Types | Societal,Economic |
Description | BigC pilot project |
Amount | £54,000 (GBP) |
Organisation | Big C Cancer Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 06/2010 |
End | 05/2011 |
Description | BigC pilot project grant |
Amount | £53,000 (GBP) |
Organisation | Big C Cancer Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2013 |
End | 09/2014 |
Description | Breast Cancer Campaign PhD studentships |
Amount | £88,000 (GBP) |
Organisation | Breast Cancer Campaign (BCC) |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2014 |
End | 09/2017 |
Description | Project research grant |
Amount | £452,800 (GBP) |
Funding ID | BB/J009040/1 |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 03/2012 |
End | 02/2015 |
Description | The BIG C PhD studentship |
Amount | £73,000 (GBP) |
Organisation | Big C Cancer Charity |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2009 |
End | 09/2012 |
Title | Generation of 3D in vitro cell cultures from various cell lines |
Description | Establishment of 3D in vitro cultures and modification of labelling protocols has enable analyses of apico-basal microtubule formation in polarising epithelial cells and of associated proteins. This work is ongoing and has further potential. |
Type Of Material | Model of mechanisms or symptoms - in vitro |
Provided To Others? | No |
Impact | The 3D cultures mimic the in vivo architecture and lateral imaging has enabled better resolution of proteins within the cells. |
Description | Advanced microscopy |
Organisation | University of East Anglia |
Department | School of Biological Sciences UEA |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Modification of some techniques, providing live samples for training and experiments |
Collaborator Contribution | Training, assistance and advise on various microscope techniques |
Impact | Training of postdoctoral researchers doi: 10.1242/jcs.129759 |
Description | End-binding proteins and breast cancer |
Organisation | Norfolk and Norwich University Hospital |
Department | Histopathology Department |
Country | United Kingdom |
Sector | Hospitals |
PI Contribution | In vitro analysis of both 2D and 3D breast epithelial cell model systems |
Collaborator Contribution | Human breast tissue samples will be prepared and analysed by a Consultant Histopathologist |
Impact | Ethical approval has been granted for samples held in the Norwich Biorepository |
Start Year | 2018 |
Description | The role of the SAS-4-related protein CPAP |
Organisation | Swiss Institute for Experimental Cancer Research (ISREC) |
Country | Switzerland |
Sector | Academic/University |
PI Contribution | Made a major contribution to this project providing extensive TEM analyses. I was a member of the supervisory team for the first author and I was also one of the external examiners for his PhD viva in Switzerland. |
Collaborator Contribution | Publication on which I was a co-author doi: 10.1016/j |
Impact | Training of postdoc Publication: doi: 10.1016/j |
Start Year | 2007 |
Description | ASCB conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Poster presentation at the American Society for Cell Biology |
Year(s) Of Engagement Activity | 2014 |
Description | American Society of Cell Biology and ECF conference, Dijon, France |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Discussions with other scientists working on microtubules and the centrosome Postdoc training in presentation of research findings |
Year(s) Of Engagement Activity | 2007 |
Description | Bio colloquium |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | Local |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Talks and poster presentations have lead to collaborations New collaborations |
Year(s) Of Engagement Activity | Pre-2006,2006,2007,2008,2009,2010,2011,2012,2013,2014 |
Description | British Society of Cell Biology meeting, Edinburgh (2009) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | National |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | poster presentation Training in presentation and networking |
Year(s) Of Engagement Activity | 2009 |
Description | Cytoskeletal conference in Potsdam Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Training and skill in presenting research findings. Stimulated new thinking and gave an opportunity to network Invitation to submit a manuscript to the journal Cell Motility and the Cytoskeleton. A manuscript was submitted, published and one of our images were selected for the front cover. doi: 10.1002 |
Year(s) Of Engagement Activity | 2008 |
Description | Cytoskeletal conference in Slovenia |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Invited speaker at the European Cytoskeletal Forum conference in Slovenia |
Year(s) Of Engagement Activity | 2015 |
URL | http://ecf2015.mf.uni-lj.si/ |
Description | EMBO conference on centrosomes, Heidelberg, Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Postdoc presented research findings as part of a poster. Postdoc awarded travel fellowship from BSCB to attend conference Training in presentation of findings postdoc wrote a meeting report on the conference which was published in the BSCB spring newsletter in 2009 |
Year(s) Of Engagement Activity | 2008 |
Description | International Cytoskeleton conference in Singapore |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | This was a major achievement and important for the development and future career of the postdoc Training and career development |
Year(s) Of Engagement Activity | 2006 |
Description | International conference on the Centrosome and Spindle Poles in Heidelberg, Germany |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | Yes |
Geographic Reach | International |
Primary Audience | Other academic audiences (collaborators, peers etc.) |
Results and Impact | Attended conference and gave a talk on our recent findings concerning the centrosome. Part of TEM work from my lab was also included on a poster by collaborator Work presented was subsequently published doi: 10.1002 doi: 10.1016/j |
Year(s) Of Engagement Activity | 2008 |