The Molecular Motors, Transport and Trafficking (M2T2) meeting serves as a platform for both Indian and global scientists working on the cytoskeleton, cytoskeletal motors and membrane trafficking to gather and discuss the latest developments in the field. The 2019 edition of the meeting, held from 18–20 October at the National Brain Research Centre (NBRC), Manesar, India and organised by Mahak Sharma (Indian Institute of Science Education and Research, Mohali) and Anindya Ghosh Roy (NBRC), was witness to stimulating research on a range of topics related to the cytoskeleton, including cytoskeletal organization, motor protein function and regulation, mechanical forces and vesicular transport, and trafficking in health and disease.
The M2T2 meeting series was conceptualized in early 2007 as a small meeting to discuss the latest trends in cytoskeletal research, with participants predominantly from India, and five subsequent meetings have been held since. What started as an informal meeting among a handful of labs involved in intracellular trafficking has grown to include tens of labs, both young and established, all interested in understanding the cytoskeleton and motor proteins in the context of intracellular trafficking.
The first M2T2 meeting was held as an informal gathering organized at the Tata Institute of Fundamental Research (TIFR, Mumbai) by Roop Mallik and Krishanu Ray (TIFR) and Debashish Chowdhury [Indian Institute of Technology (IIT) Kanpur]. Subsequent meetings, organized roughly biennially, have been nucleated by Roop Mallik, Krishanu Ray and Sandhya Koushika (TIFR), and coorganized in different editions by Gautam Menon (Ashoka University), Dulal Panda and Ambarish Kunwar (IIT Bombay, Mumbai) and Subhojit Roy (University of California, San Diego). In the past decade, the number of researchers in India investigating cytoskeleton-mediated processes has seen a steady increase. The M2T2 meetings have been instrumental in bringing this growing pool of researchers together to discuss the newest developments, share ideas and resources, and forge collaborations.
Here, we collate the proceedings of the most recent edition of the M2T2 meeting, organized by Mahak Sharma (Indian Institute of Science Education and Research, Mohali) and Anindya Ghosh Roy [National Brain Research Centre (NBRC), Manesar, India] and held at the NBRC from 18–20 October, 2019. The meeting featured invited talks by principal investigators, as well as selected postdoctoral fellows and graduate students. The sessions were categorized into multiple themes, as summarized below.
Cytoskeletal organization and organelle positioning in neurons
The organization of the cytoskeleton and organelles in neurons is central to the ability of a neuron to receive, process and transmit information. Melissa Rolls (Pennsylvania State University, State College, USA) kicked off the meeting by discussing the use of microtubule minus-end-out dendrites in Drosophila neurons to study the control of microtubule polarity. She proposed that kinesin-5 acts with another protein to allow newly nucleated microtubules to grow along existing microtubules oriented in the same direction, thus creating a positive-feedback loop to promote uniform microtubule polarity in dendrites.
Aurnab Ghose (Indian Institute of Science Education and Research, Pune) then presented the current work of his laboratory, which reveals that the formin family member Fmn2 regulates growth cone translocation by modulating a force-generating ‘molecular clutch’ at adhesive contacts between the growth cone and the extracellular matrix. Having previously identified Fmn2 as a regulator of axon pathfinding, Aurnab additionally described a novel actin-microtubule crosslinking activity of Fmn2 that regulates microtubule dynamics in the growth cone (Kundu et al., 2020 preprint).
Mitochondria are essential both for providing ATP to power nerve cell function and for Ca2+ buffering. Sandhya Koushika (Tata Institute for Fundamental Research, Mumbai, India) discussed findings from her lab that, in individual C. elegans touch receptor neurons, mitochondrial positions appeared to be regulated by a cell intrinsic co-incidence detection mechanism of actin coupled with local Ca2+ chemical signals. Swagata Dey (National Brain Research Centre, Manesar, India) then addressed the regeneration of axon and dendrites upon injury and the concomitant cytoskeletal changes that accompany the process. Her work showed that axon injury causes a retraction of the severed end followed by regrowth from the severed end, or remodeling of the neighboring neurites. She intends to further explore cytoskeletal regulators that can act as signaling effectors.
Taken together, the speakers of this session answered important questions relating to the organization and dynamics of the cytoskeleton in neurons and its functional role in determining the positioning of mitochondria and axonal regeneration upon injury.
Mitosis – microtubule dynamics, motors and spindle organization
Mitosis requires the concerted activity of microtubules and motor proteins to bring about spindle formation, chromosome alignment and finally, segregation. Sachin Kotak (Indian Institute of Science, Bengaluru) discussed the balance between protein phosphorylation and dephosphorylation in regulating the appropriate orientation of the mitotic spindle, which is required for error-free cell division. He described a candidate-based chemical genetics approach to discover a novel pathway by which Cdk1 and its counteracting B55-containing PP2A phosphatase complex orchestrate the spatiotemporal levels of the cortical NuMA–dynein complex for flawless spindle behavior.
The lack of proper kinetochore–microtubule coupling leads to chromosome loss and aneuploidy, which are hallmarks of cancer in humans. Dileep Varma (Northwestern University, Evanston, USA) spoke about the mechanisms that control stable attachments between kinetochores and spindle microtubules during mitotic chromosome alignment and segregation. The work presented provided evidence for a synergy between multiple kinetochore microtubule-associated proteins (MAPs) that enable the effective coupling of the Ndc80 complex to dynamic microtubule plus-ends during metaphase (Campbell et al., 2019).
Chandan Kumar (Regional Centre for Biotechnology, Faridabad, India) discussed efforts to unravel the function of a novel dynein-interacting protein in achieving chromosome alignment at metaphase, spindle pole integrity and progression to anaphase. Interestingly, the mitotic interactome of this protein includes proteins of the endocytic trafficking pathway. The ongoing study is likely to reveal novel molecular mechanisms that regulate mitotic progression in human cells.
These talks provided novel molecular mechanistic insight into the orientation of the mitotic spindle within the cell, chromosome capture by pole-derived microtubules preceding segregation, and the potential novel interplay between a dynein interactor and endocytic traffic.
Microtubule–motor interactions – different modes of regulation
Motor proteins exhibit a range of behaviors on their cytoskeletal tracks, which not only depend on their inherent properties, but also on the track itself, such as any post-translational modifications of tubulin. Chaitanya Athale (Indian Institute of Science Education and Research, Pune) examined emergence of coordinated transport in models of microtubule polarization during neuronal growth cone guidance, meiotic acentrosomal spindle assembly and in a microtubule gliding assay. He argued that the presence of emergent behavior in cooperative polarization, assembly and transport suggests that collective properties of microtubule–motor mixtures could have a functional role in vivo (Jain et al., 2019). Nitin Mohan (Indian Institute of Technology, Kanpur) then presented data obtained by correlative live-cell and super-resolution STORM to show that detyrosinated microtubules facilitate efficient fusion between autophagosomes and lysosomes, and depletion of detyrosinated microtubules leads to a significant reduction in autophagy (Mohan et al., 2019).
Mitochondrial fission is orchestrated by the dynamin-related protein Dnm1, and cells devoid of Dnm1 exhibit a single large mitochondrion. Building on past research (Mehta et al., 2019), Leeba Ann Chacko (Indian Institute of Science, Bengaluru) now showed that perturbing mitochondrial morphology disturbs cell polarity and symmetry during cell division in S. pombe, likely by interfering with normal microtubule dynamics in these cells.
Sukriti Kapoor (Indian Institute of Science, Bengaluru) investigated the role of centrosome enriched Aurora A kinase (AIR-1) in polarity establishment in the one-cell stage of the C. elegans embryo. She showed that depletion of AIR-1 results in the impairment of cortical flows and leads to the formation of an ectopic posterior PAR domain. Furthermore, she discovered that the Rho-GEF ECT-2 acts downstream of AIR-1, and that co-depletion of ECT-2 and AIR-1 rescues the bipolar polarity defects observed upon AIR-1 loss (Kapoor and Kotak, 2019).
The speakers of this session demonstrated distinct instances of interplay between motor proteins, organelles and microtubules that resulted in coordinated transport, fusion of endosomal compartments, cell division symmetry and polarity establishment.
Location, location, location – cargo positioning by motors
The dynamic positioning of organelles and other cargo by motor proteins affects complex cellular functions, including development and differentiation. Vladimir Gelfand (Northwestern University, Evanston, USA) described the mechanisms for positioning of the posterior-specific mRNA marker oskar (osk), along with its binding protein Staufen in the Drosophila oocyte. He demonstrated that posterior localization of osk–Staufen is determined by competition between the kinesin-1 and myosin-V motors. Whereas kinesin-1 removes osk–Staufen from the cortex along microtubules, myosin-V anchors osk–Staufen at the cortex. Thus, posterior determination in the Drosophila oocyte is defined by a competition between kinesin and myosin, whose outcome is primarily determined by cortical microtubule density (Lu et al., 2020).
Ambarish Kunwar (Indian Institute of Technology Bombay, Mumbai) discussed cargo positioning mediated by a team of motor proteins. He first showed that temperature differently affects the biophysical properties of kinesin and dynein, suggesting that temperature could potentially be used as a non-invasive probe of intracellular transport. Next, he described a recently developed experimental technique named ‘reversible association with motor proteins’ (RAMP) to manipulate the position of various types of cargo within the cytoplasm (Guardia et al., 2019). Finally, he discussed a computational model to predict experimentally observed trends of the peri-nuclear or peripheral accumulation of cargos.
Roop Mallik (Tata Institute of Fundamental Research, Mumbai) presented new mechanisms and potential targets in lipoprotein secretion from the liver. Building on their own earlier work showing that kinesin transports triglyceride-rich lipid droplets (LDs) to the smooth ER in liver cells, he described that triglycerides are then packaged into VLDL-containing particles and secreted from the liver into the blood. This pathway is controlled by insulin, and therefore responds to the fed or fasted state of the animal (Kumar et al., 2019a).
This set of talks emphasized the diversity of mechanisms employed by molecular motors and their mutual interplay in deciding important cellular functions, and suggested that there is a wealth of knowledge to be uncovered about the functioning of intracellular motor proteins in various contexts in health and disease.
Mechanical forces at play – guiding cellular morphology and migration
Mechanical forces, exerted by the cytoskeleton in concert with motor proteins, are required for a variety of cellular processes, including cell division, development, migration and, in general, proper functioning of cells. Pramod Pullarkat (Raman Research Institute, Bengaluru, India) combined measurements obtained from biochemical and genetic modifications to the axonal cytoskeleton with those obtained using a custom-built force apparatus to explore the mechanical response of axons. Using a theoretical model, he argued that the actin–spectrin skeleton acts as an axonal tension buffer of mechanical stress by reversibly unfolding repeat domains of the spectrin tetramers.
Collective cell dynamics refers to the process of many cells acting as a cohesive group, with each individual adjusting and synchronizing its dynamics with that of its neighbors. Tamal Das (Tata Institute of Fundamental Research, Hyderabad, India) addressed how mechanical forces exerted at cell–cell and cell–matrix junctions influence the dynamics at multicellular length-scales, and how this dictates the process of intracellular reorganization during cell competition. His talk revealed the underlying biophysical dynamics, structural reorganization, and molecular mechanisms that enable cell collectives to forcibly remove a malignantly transformed cellular population.
Semiflexible filaments driven by motor proteins constitute a situation prevalent in the cytoskeleton, and are often studied in simplified molecular motor assay setups. Debashish Chaudhuri (Institute of Physics, Bhubaneswar, India) described an explicit model involving a gliding assay of motor proteins driving semiflexible filaments, which show non-equilibrium ring formation and a series of interspersed diffusive and directed movements. In the context of the mitotic spindle, he postulated that the presence of diffusing cross-linker proteins may generate contractile overlap between filaments, which would compete against extensile pulls of motor proteins, thereby leading to dynamical phases depending on the degree of activity (Gupta et al., 2019).
These talks on the physical properties of the cytoskeleton and other proteins, which affect both individual and collective cell behavior, suggested that further studies on the biophysical attributes of the cytoskeleton would reveal novel aspects of the regulation of cell behavior and function.
Monitoring the molecular motor
The activity of motor proteins is typically regulated in living cells, such as by autoinhibition of the motor until it binds to cargo. Recent in vitro studies (McKenney et al., 2014; Schlager et al., 2014) have shown that the major minus-end-directed motor protein cytoplasmic dynein 1 (henceforth dynein) requires its regulator dynactin, and a cargo adaptor for activity. In her talk, Vaishnavi Ananthanarayanan (Indian Institute of Science, Bengaluru) addressed the question of how and where dynein is activated inside living cells by employing single-molecule imaging of dynein in vivo.
Within a cell, bidirectional transport of cargo on microtubule filaments is brought about by oppositely directed motor proteins, such as dynein and kinesin. However, cells often show the so-called ‘paradox of codependence’, where there is an overall decline of motility of the cargo upon inhibition of one motor species. Abhishek Chaudhuri (Indian Institute of Science Education and Research, Mohali) presented a possible resolution for this paradox when the catch-bonding behavior of dynein is incorporated into a theoretical model (Puri et al., 2019).
By employing single-molecule imaging of motor proteins in vivo and mathematical modeling of motor behavior, the speakers of this session thus addressed how motor regulation occurs in living cells.
Mechanism of cytokinesis
Cytokinesis, the terminal step of cell division, is brought about by a complex series of events involving primarily the actin cytoskeleton and motor proteins. Mohan Balasubramanian (Warwick Medical School, Warwick, UK) described long term efforts of his group to use minimal systems to understand eukaryotic cytokinesis by using the fission yeast, which divides using an actomyosin contractile ring, as a model organism. He presented evidence for the division of fission yeast protoplasts, as well as results from in vitro reconstitution experiments, which showed that contractile actomyosin rings are assembled from five proteins embedded on a supported lipid bilayer.
Sivaram Mylavarapu (Regional Centre for Biotechnology, Faridabad, India) described a recent study from his group that unexpectedly unearthed an interaction between the conserved exocyst complex with the early endocytic GTPase Rab5 in cytokinetic cells. Both Rab5 and exocyst complex subunits localize at the midbody ring in the cytokinetic bridge and are required for delivering the membrane abscising ESCRT-III machinery to this site to enable abscission. This work suggests that early endosomes could play roles in cytokinetic abscission in addition to the documented roles of secretory vesicles and recycling endosomes (Kumar et al., 2019b).
This set of talks reaffirmed the complexity of the various cellular processes that need to be orchestrated to ensure the fidelity of the final steps that culminate in the generation of two daughter cells, and suggested that this remains a fertile area for future exploration.
Cell biology of pathogens and their hosts
During their infection cycle, pathogens exploit the host intracellular trafficking machinery to traverse the cell for replication, or to exit the host, and these aspects were covered in two sessions at the meeting. Rupinder Kaur (Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India) started the first session on this theme with a discussion of the role for phosphoinositide 3-kinase (PI3K) in the vesicular trafficking and pathogenesis of Candida glabrata. Her talk implicated C. glabrata (Cg)Vps34 in the vesicular trafficking, autophagy, iron homeostasis, biofilm formation and virulence of the organism, in addition to retrograde trafficking of metal ion transporters from the plasma membrane in response to environmental cues (Sharma et al., 2016).
Sheetal Gandotra (CSIR Institute of Genomics and Integrative Biology, New Delhi, India) presented data on fatty acid flux between host macrophages and Mycobacterium tuberculosis (Mtb), and the contribution of the macrophage LDs in this phenomenon. Her group had demonstrated that the bacteria compete with the host lipid-storage machinery. To understand whether bacteria modify LDs, they undertook a proteomics approach, which revealed that the localization of the small GTPase ARL8B to LDs is actively manipulated by the bacteria, suggesting that this engagement of ARL8B may be important during infection (Menon et al., 2019).
Intracellular pathogens commonly manipulate the host endo-lysosomal system. However, it is not known whether this affects the organization and functioning of the endo-lysosomal system. Varadharajan Sundaramurthy (National Centre for Biological Sciences, Bengaluru, India) showed that the endo-lysosomal system is globally elevated in Mtb-infected macrophages. He showed that lysosomes in Mtb-infected cells are modulated by mycobacterial factor(s), predominantly the sulfolipid SL-1, which induces lysosomal biogenesis by modulating the host mTORC1–TFEB axis. The elevated number of lysosomes functions to restrict intracellular Mtb survival (Sachdeva et al., 2019 preprint).
The second session on this theme commenced with a talk by Manjula Kalia (Regional Centre for Biotechnology, Faridabad, India), who discussed membrane-trafficking networks exploited by the Japanese encephalitis virus (JEV). JEV endocytosis into HeLa cells is largely dependent on components of clathrin-mediated endocytosis. Her talk also highlighted the common themes, as well as key differences, in the membrane trafficking requirements of JEV between epithelial and neuronal cell lineages (Khasa et al., 2019). In his presentation, Bhavani S. Sahu (National Brain Research Centre, Manesar, India) focused on organellar proteomics as a tool to understand the role of clathrin in dense core vesicle (DCV) biogenesis. By using subcellular fractionation with quantitative mass spectrometry, he was able to show that clathrin has a function beyond its established endocytic role in removing unwanted proteins from the immature vesicle during DCV biogenesis (Sahu et al., 2017).
Shailja Singh (Jawaharlal Nehru University, New Delhi, India) described her work on the role of signaling and cytoskeletal rearrangements in Plasmodium falciparum exocytosis and the regulation of infection and pathogenesis. She demonstrated that the parasite tubulin orchestrates the discharge of micronemes to establish infection, while, in turn, cytosolic Ca2+, cAMP and phosphatidic acid homeostasis govern the discharge of micronemal contents, such as perforin-like protein-1 (Garg et al., 2020).
The various talks in this session underscored the importance of intracellular trafficking pathways in the pathogenicity of a variety of microbial pathogens. The central theme highlighted by these studies was the evolution of pathogenic factors, which try to hijack host cell pathways for their own benefit, perhaps also throwing open potential areas of intervention from a therapeutic standpoint.
The M2T2 2019 meeting was a congregation of international and local scientists with a shared passion for research on the cytoskeleton, molecular motors and membrane traffic. The collection of scientific talks and ensuing discussions underscored the importance of studying intracellular cytoskeletal organization and dynamics, and motor-based intracellular transport as well as vesicular trafficking, inter-related fields of research with intricate relationships to health and disease states. Despite the intense ongoing work globally, a plethora of questions remains to be addressed in these areas, with the potential of being exploited for therapeutic benefit. In addition to the scientific program, there was a panel discussion with Vladimir Gelfand, Mohan Balasubramanian, Roop Mallik, Melissa Rolls and Vaishnavi Ananthanarayanan on ‘Scientific Publishing’. The topics discussed ranged from how to choose a journal for publishing your work, Plan S compliance and effective peer review. The meeting concluded with a summary by organizers Mahak Sharma and Anindya Ghosh Roy. We look forward to the 2021 edition of the meeting, which will be held in Bengaluru and organized by Sachin Kotak and Vaishnavi Ananthanarayanan.
The authors thank Roop Mallik, Krishanu Ray, Sandhya Koushika, Mahak Sharma and Anindya Ghosh Roy for inputs and comments on the report. The M2T2 2019 meeting was supported by the following organizations: National Brain Research Centre (NBRC), Manesar, India; Indian Institute of Science Education and Research (IISER) Mohali, India; Department of Biotechnology, Ministry of Science and Technology, Government of India; The Company of Biologists, Cambridge, UK; Towa Optics India Pvt. Ltd.; Nikon Corporation, Japan; Merck Group, Sigma Aldrich Chemicals Pvt. Ltd.; Eppendorf India Pvt. Ltd. and SciMed (Asia) Pte Ltd., Panasonic Health Care Biomedical". V. A. is supported by extramural funding from the Wellcome Trust/Department of Biotechnology–India Alliance (grant IA/18/1/503607), and the Women Excellence Award from the Science and Engineering Research Board (SERB), India, and intramural funding from the Indian Institute of Science, and the RI Mazumdar Young Investigator Award. S. V. S. M. is supported by extramural grants from SERB, India and intramural funding from the Regional Centre for Biotechnology, India.
The authors declare no competing or financial interests.