A biophysical study on how the actin and microtubule cytoskeletons dynamically collaborate to regulate cellular organization

Lead Research Organisation: University of Cambridge
Department Name: Zoology

Abstract

A fundamental question in biology is how complex animals originate from the first cell, the egg. Embryologists suggested long ago that the asymmetric distribution of substances, called determinants, in the cytoplasm of an egg (known as egg polarization) could confer a specific characteristic to the cells that receive them upon cell division. One of the most striking examples of egg polarization is found in Drosophila, in which the asymmetric localization of various determinants during oogenesis establishes the body plan. For these developmental determinants to localize properly, a variety of complex processes need to take place. Firstly, cells (such as the egg) are not symmetric entities; rather they have to adopt particular asymmetries, which are critical to define the final position of the determinants. Additionally, the fluid through which the determinants navigate, known as cytoplasm is not quiescent. Instead it is a swirling, jetting, dynamic fluid. Thus, to understand how the asymmetric distribution of substances is achieved, we need to study not only the movement of the substance, but also the motion of the highly dynamic cytoplasm that surrounds the substance.

The link between the movement of molecules and the fluid dynamics of the cytoplasm is not only a theoretical one, but also a mechanistic one. Cells have two complex filamentous structures, known as the actin and microtubule (MT) cytoskeletons that exert forces and drive transport of molecules and organelles. These cytoskeletons, and a group of proteins (known as molecular motors) that "walk" along them, are required to transport determinants, but also to induce cytoplasmic flows. At times actin, MTs and their motor proteins act as separate systems, but there is also often poorly understood crosstalk between the two cytoskeletons. In the Drosophila female germline, localization of developmental determinants is governed by the interplay among actin, MTs and the motor protein Kinesin. Both directed transport and cytoplasm streaming, are mediated by this interplay. This constitutes a powerful cellular organization model of developmental importance in which the two cytoskeletons dynamically modulate each other.

We will employ a unified theoretical and experimental approach to understand how the actin and MT cytoskeletons coordinate the essential, and developmentally regulated, asymmetries and motions during oogenesis. Further, we will explore how streaming impacts on the organization of the cytoskeletons, and what flows reveal about the underlying forces. Specifically, we will investigate: 1. The bi-directional link between cytoplasmic flows and the MT cytoskeleton; and 2. The regulation of streaming, MTs and Kinesin by the actin cytoskeleton.

Technical Summary

Cellular asymmetries strongly rely on actin and microtubule (MT) cytoskeletons. At times these act as separate systems, but there is often poorly understood crosstalk between the two cytoskeletons. During Drosophila midoogenesis, developmental determinants are transported on MTs. The action of the motor Kinesin on MTs also induces cytoplasmic streaming. It is hypothesized that cytoplasmic F-actin regulates both processes, but the nature of the crosstalk between actin and microtubules and the interplay between streaming and the cytoskeletons remain largely unknown. Flows, actin and MTs not only interact during mid-oogenesis, when the oocyte is highly polarized, but also in late oogenesis, when the cell undergoes a rapid reorganization. The project focuses on using biophysics to study the bi-directional interaction between the cytoplasm fluid dynamics and the cytoskeletons, using tools of quantitative imaging, theoretical fluid dynamics, microfluidics and genetics. We believe the oocyte to be a powerful cellular organization model in which the two cytoskeletons dynamically modulate each other: How the two cytoskeletons interact with each other in regulating cellular organization is an area of increasing importance.

Our aims are to: i) Study the relation between motors, viscosity and flows. In vivo work will be complemented by in vitro assays to test the biophysical relation between the force generating entities and flows; ii) Analyze the correlation between flows and two movements of the MTs: the bulk arrangements of bundles, and the dynamics of the plus-ends; iii) Analyze whether the actin mesh regulates flows, kinesin and MT organization. We will also characterize the biophysical features of the actin mesh, and how they correlate with those of streaming.

This proposal fits well into the priority 'systems approach to biological research' by using experimental and theoretical physics to study a complex system determined by the properties of many moving parts

Planned Impact

This proposal will contribute to the understanding of basic cellular mechanisms that are essential for cell organization and cell asymmetries. This contribution is also important for human health for several reasons. Since the cytoskeleton is involved in virtually all cellular processes, including cell proliferation, abnormalities in this cellular component frequently result in disease. Also, there is some evidence that the defective function of motors results in various human diseases, such as Alzheimer and retinitis pigmentosa. Kinesin is specifically linked to Charcot-Marie-Tooth disease and Hereditary Spastic Paraplegia. In addition, the asymmetric localization of molecules is important not only in the establishment of the mammalian body plan, but also in the function of most cells, including neurons, where it is involved in learning and is defective in various diseases. The application explores a fundamental problem at the interface between biology and physics, and thus has a potential transformative capability, which affect many fields. Also, this approach may help create a new field of biophysics, so its long-term impact could be very high. The strong contribution of this research to medicine would have an positive effect on the competitiveness of the economy and would lead to improvement of our quality of life.

Our research will also generate benchmark observations, which will connect with growing interest within the fluid dynamics community in the behavior of systems composed of many motile components. A considerable amount of recent research in this general area suggested interesting potential applications in autonomous robotics and microfluidics.

Exploitation: Although this proposal mainly contributes to the understanding of basic cellular mechanisms, it is possible in an unpredictive manner that it leads to new ways of preventing abnormal cell behavior and human diseases (e.g., through the development of new drugs). Other areas for possible exploitation include the field of microfluidics, where mixing on the micron scale is a longstanding challenge. If this were the case, the University of Cambridge has extensive experience of technology transfer through their company Cambridge Enterprise

Outreach: Our results will be presented at a wide range of interdisciplinary scientific meetings worldwide, and at various EU summer schools/workshops, which Goldstein co-directs in the areas of biological physics. Both principal investigators are linked to various outreach associations. These are: The Cambridgeshire Branch of the British Science Association; The Cambridge Association for Women in Science and Engineering (AWiSE), The Millennium Maths Project, and public lectures through the Institute of Physics and the Royal Institution. In addition, both PIs plan to contribute in the next few years to the Cambridge Science Festival (Goldstein has previously participated), one of the largest in the country http://comms.group.cam.ac.uk/sciencefestival/. As a former URF, Palacios is in continuous contact with the Royal Society, which influences policymaking with its scientific advice and invigorates science education. The Department of Applied Mathematics and Theoretical Physics regularly has Open Days in which hundreds of members of the public view a wide range of research activities in areas as diverse as fluid dynamics, particle physics, and cosmology

Skills:There are various skills that the staff working on the project will develop which they could apply to other sectors. These are: project management; definition and achievement of milestones; efficiently working within a group; successfully deal with deadlines; designing/performing/analyzing experiments;communication skills both in writing and in oral presentations to worldwide peers; and commercial awareness. The fundamental interdisciplinary character of the research provides the opportunity to improve communication skills among a very diverse group of scientists

Publications

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Palacios IM (2014) Hop-on hop-off: polysomes take a tour of the cell on endosomes. in The Journal of cell biology

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Gómez-Lamarca MJ (2014) Integrins regulate epithelial cell differentiation by modulating Notch activity. in Journal of cell science

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Palacios IM (2013) Nonsense-mediated mRNA decay: from mechanistic insights to impacts on human health. in Briefings in functional genomics

 
Description Cytoskeletal networks do not exist in isolation, but experience crowded and dynamic intracellular environments. However, microtubule-environment interactions are not well understood, and such system-environment interactions are an unresolved question in biology that demands bridging across disciplines. Here we introduce an Optical Flow motion estimation approach to study microtubule orientation in the Drosophila oocyte, a cell displaying substantial cytoplasmic streaming. We show that microtubule polarity is affected by the regime of these flows, and furthermore, that the presence of flows is necessary for MTs to adopt their proper polarity. With these findings we are contributing to further understanding how microtubules organize in their impacting natural environment.
Exploitation Route The orientation of microtubule networks is exploited by motors to deliver cargoes to specific intracellular destinations, and is thus essential for cell polarity and function. Reconstituted in vitro systems have largely contributed to understanding the molecular framework regulating the behavior of microtubule filaments. In cells however, microtubules are exposed to various biomechanical forces that might impact on their orientation, but little is known about it. Oocytes, which display forceful cytoplasmic streaming, are excellent model systems to study the impact of motion forces on cytoskeletons in vivo. Here we implement variational optical flow analysis as a new approach to analyze the polarity of microtubules in the Drosophila oocyte, a cell that displays distinct Kinesin-dependent streaming. After validating the method as robust for describing microtubule orientation from confocal movies, we find that increasing the speed of flows results in aberrant plus end growth direction. Furthermore, we find that in oocytes where Kinesin is unable to induce cytoplasmic streaming, the growth direction of microtubule plus ends is also altered. These findings lead us to propose that cytoplasmic streaming - and thus motion by advection - contributes to the correct orientation of MTs in vivo. Finally, we propose a possible mechanism for a specialised cytoplasmic actin network (the actin mesh) to act as a regulator of flow speeds: to counteract the recruitment of Kinesin to microtubules.
Sectors Education,Healthcare,Pharmaceuticals and Medical Biotechnology

URL https://palacioslabqmul.squarespace.com/
 
Description 2021 Drosafrica workshop online Ghana University 2019 Drosafrica workshop online, plus attending and co-organising the first Annual Meeting of the African Drosophila Research Society (ADRS) in Ibanda, Nigeria 2017 Organiser and Lecturer, DrosAfrica workshop: Drosophila as a model for human diseases: Affordable and Impacting at the University of Ibadan, Nigeria. Since 2016 Ambassador in Cambridge for the British Society of Cell Biology Since 2016 Member of the ASCB International Affairs Committee 2016 Organiser and Lecturer: DrosAfrica workshop: Drosophila as a model for human diseases: Affordable and Impacting, ICIPE, Nairobi, Kenya 2015 Member of the DFG Grants Committee, Germany. 2015 Organizer of the 3rd International Symposium SRUK, London Since 2014 Member of the Society of Spanish Researchers in the UK (SRUK) Since 2014 Academic Editor of PeerJ 2013-2015 Organiser and Lecturer: DrosAfrica Developmental Genetics workshop, Kampala International University, Uganda 2013 Founder of the charity DrosAfrica. Helping to set up an African Drosophila research community (see http://drosafrica.org for details) 2011-13 Lecturer at the Trend/IBRO Drosophila Neurogenetics course in Kampala International University, Uganda 2010-14 Member of the Grants Committee for the Agence Nationale De La Recherche (ANR), France
First Year Of Impact 2019
Sector Education,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal,Economic

 
Description We have created new labs using Drosophila as a model system in Africa, in countries where there were no model system labs set up.
Geographic Reach Africa 
Policy Influence Type Influenced training of practitioners or researchers
 
Description ARUK Pilot Project
Amount £43,570 (GBP)
Funding ID ARUK-PPG2015B-7 
Organisation Alzheimer's Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 03/2016 
End 03/2017
 
Description start up package
Amount £90,000 (GBP)
Organisation Queen Mary University of London 
Sector Academic/University
Country United Kingdom
Start 08/2017 
 
Title DDM as a method to analyse the type of motion of cellular components. An extremely powerful and efficient manner to distinguish various type of motions, in a non-invasive way 
Description DDM as a method to analyse the type of motion of cellular components. An extremely powerful and efficient manner to distinguish various type of motions, in a non-invasive way 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? Yes  
Impact Any biologist, or physicist that needs to understand intracellular motion, or cellular motion, in any healthy or diseased state, will be able to benefit from this finding 
 
Title PIV 
Description Demonstrate that PIV can be used ain the cytoplasm of the oocyte to measure fluid dynamics 
Type Of Material Biological samples 
Year Produced 2012 
Provided To Others? Yes  
Impact
 
Title transgenics and mutants 
Description Creation of many transgenes and mutations in genes in pathways that are conserved in humans, form the motor protein Kineisn to components of the Hippo pathway 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Year Produced 2007 
Provided To Others? Yes  
Impact further understanding of cancer cell biology 
 
Title The source code of our implementation and of the data analysis is available online (https://doi.org/10.5281/zenodo.2573254). All relevant data and the computational results are available from the corresponding authors upon request. 
Description The source code of our implementation and of the data analysis is available online (https://doi.org/10.5281/zenodo.2573254). All relevant data and the computational results are available from the corresponding authors upon request. 
Type Of Material Computer model/algorithm 
Year Produced 2019 
Provided To Others? Yes  
Impact The source code of our implementation and of the data analysis is available online (https://doi.org/10.5281/zenodo.2573254). All relevant data and the computational results are available from the corresponding authors upon request. 
URL https://www.biorxiv.org/content/10.1101/556043v3
 
Description Collaboration with Dr. Livesey 
Organisation University of Cambridge
Department Gurdon Institute
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution My postdoc and I together with Dr. Livesey's postdoc have been analysing the effect on neurodegeneration when reducing or increasing kinesins levels. Our findings in Drosophila have been so far confirmed in iPS-derived neurons
Collaborator Contribution My postdoc and I together with Dr. Livesey's postdoc have been analysing the effect on neurodegeneration when reducing or increasing kinesins levels. Our findings in Drosophila have been so far confirmed in iPS-derived neurons
Impact The project is importing findings in an invertebrate, to human neurons. This approach allows for a fast finding of the molecular basis of neurodegeneration in Drosophila, which can then be tested in human neurons.
Start Year 2016
 
Description Collaboration with Dr. Roberto Cerbino's lab 
Organisation University of Milan
Country Italy 
Sector Academic/University 
PI Contribution My postdoc and I collaborated closely with the physicists on the motions of cytoplasmic components. Results in a publication. Currently preparing another manuscript and a new grant
Collaborator Contribution We contributed with the biological aspect of the system They contributed with the physics and mathematical aspect of the system.
Impact This is a truly interdisciplinary collaboration
Start Year 2015
 
Description Collaboration with MD Martin Bermudo lab 
Organisation Andalusian Center for Development Biology
Country Spain 
Sector Private 
PI Contribution We have analysed the role of Spectrins, Integrins, Notch and Hippo in the follicular epithelium of the Drosophila germline
Collaborator Contribution They have analysed the role of integrins in the follicular epithelium of the Drosophila germline
Impact Two publications Gómez-Lamarca MJ, Cobreros-Reguera L, Ibáñez-Jiménez B, Palacios IM and Martín-Bermudo MD. (2014). Integrins regulate epithelial cell differentiation by modulating Notch activity. J. Cell Science Ng BF, Selvaraj GK, Santa-Cruz Mateos C, Grosheva I, Alvarez-Garcia I, Martín-Bermudo MD and Palacios IM (2016). Alpha-Spectrin and Integrins act together to regulate actomyosin and columnarization, and to maintain a mono-layered follicular epithelium. Feb 2016, accepted in Development.
Start Year 2016
 
Description Collaboration with Peter Lawrence lab 
Organisation University of Oklahoma
Department Department of Zoology
Country United States 
Sector Academic/University 
PI Contribution we shared a PhD student that resulted in two publications. We contributed intelectually and financially Saavedra P, Vincent JP, Palacios IM, Lawrence PA and Casal J (2014). Plasticity of both planar cell polarity and cell identity during the development of Drosophila. eLife, February 11, 2014;3:e01569. Pedro Saavedra, Amy Brittle, Isabel M. Palacios, David Strutt, José Casal and Peter A. Lawrence (2016). Planar cell polarity: the Dachsous-Fat system contributes differently to the embryonic and larval stages of Drosophila. Accepted in Biology Open.
Collaborator Contribution My partners contributed both intelectually and by performing experiments, as well as financially
Impact Saavedra P, Vincent JP, Palacios IM, Lawrence PA and Casal J (2014). Plasticity of both planar cell polarity and cell identity during the development of Drosophila. eLife, February 11, 2014;3:e01569. Pedro Saavedra, Amy Brittle, Isabel M. Palacios, David Strutt, José Casal and Peter A. Lawrence (2016). Planar cell polarity: the Dachsous-Fat system contributes differently to the embryonic and larval stages of Drosophila. Accepted in Biology Open. https://www.repository.cam.ac.uk/handle/1810/253638?show=full
Start Year 2009
 
Description Collaboration with Prof Ray E Goldstein, physicists 
Organisation University of Cambridge
Department Department of Applied Mathematics and Theoretical Physics (DAMTP)
Country United Kingdom 
Sector Academic/University 
PI Contribution My PhD student and I collaborated closely with the physicists on the fluid dynamics of the oocyte cytoplasm. resulting in a publication. We contributed with the biological aspect of the system
Collaborator Contribution They contributed with the physics and mathematical aspect of the system. This is a truly interdisciplinary collaboration
Impact A publication A BBSRC grant
Start Year 2009
 
Description First ever conference of the African Society of Drosophila Research 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Our research has contributed to the creation of the first African Society of Drosophila Research. We then contributed to their first conference, in Ibadan, Nigeria.
Year(s) Of Engagement Activity 2019
 
Description Founder of DrosAfrica, higher Education 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact 2011-13 Lecturer at the Drosophila Neurogenetics course in Kampala International University, Uganda
2013-2015 Organiser and Lecturer: DrosAfrica Developmental Genetics workshop for researchers in Africa, Kampala International University, Uganda
2013 Founder of DrosAfrica. Setting up Drosophila labs in Africa, and organising workshops to create an African Drosophila research
community (see http://drosafrica.org for details and press)
2017 Organiser and Lecturer: DrosAfrica course: Drosophila as a model for human diseases: Affordable and Impacting, University of Ibadan, Nigeria
2016 Organiser and Lecturer: DrosAfrica course: Drosophila as a model for human diseases: Affordable and Impacting, two workshops: one in September at ICIPE, Nairobi, Kenya, and one in October at University of Jos, Nigeria.

Baden T, Palacios I, Vicente M, Martin-Bermudo D, Yusuf S, Prieto Godino L (2012). Higher Education in the Developing World: Drosophila Neurogenetics, Uganda. INTED2012, Valencia, Spain.

Baden T, Palacios I, Vicente M, Martin-Bermudo D, Yusuf S, Prieto Godino L (2011). Breaking the Wall of Global Inequality in Higher Education. Falling Walls Conference, Berlin, Germany.
Year(s) Of Engagement Activity 2011,2012,2013,2014,2015,2016
URL http://drosafrica.org
 
Description Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact A workshop at International Day at QMUL
Year(s) Of Engagement Activity 2018
 
Description Workshop at Sfax University in Tunisia 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact This workshop has now led to a longterm relationship with Tunisia, involving the Ministry of Education, and various North African Universities
Year(s) Of Engagement Activity 2019
 
Description international research conferences 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Members of my lab and myself attend regular national and international conferences where we present research talks, or research posters. We attend an average of 4-5 meetings or conferences a year
Year(s) Of Engagement Activity 2017,2018,2019,2020