Postdoctoral Program - Joint projects
JOINT PROJECT NO. 1
Mechanistic Understanding of Clathrin and Dynamin-Independent Endocytic Pathways
It is evident that several endocytic mechanisms exist for the internalization of extracellular or plasma membrane proteins, and that are necessary for nutrient uptake, cell signaling and infection. While the importance of clathrin and dynamin-dependent mechanisms is well-established, the roles of clathrin and dynamin-independent mechanisms is only now being recognised. The laboratories of Satyajit Mayor at the National Centre for Biological Science (TIFR) (NCBS-TIFR, Bangalore, India) and Ludger Johannes at Institut Curie (IC, Paris, France) have uncovered new molecular machinery for these processes . While the Mayor laboratory has focused on the uptake of GPI-anchored proteins via the CLIC/GEEC pathway, and the Johannes laboratory on bacterial and cellular lectins such as Galectin-3 and their interactions with glycosphingolipids and glycosylated cargo proteins via the GlycoLipid-Lectin (GL-Lect) mechanism. In the current joint program between NCBS and IC, we wish to use state-of-the art live cell imaging (such as lattice light sheet microscopy at IC, TIRF combined with pH pulsing and fluorescence emission anisotropy imaging at NCBS) and advanced cellular biochemistry techniques (such as DNA nanotechnology-based endocytic carrier purification) for the molecular dissection of the mechanisms that lead to the clathrin-independent formation of endocytic pits responsible for the uptake of these cargo. We expect to uncover general principles that would be common to mechanisms for building endocytic pits without involvement of the clathrin and dynamin machinery. The mechanism of uptake of these cargo will also provide insights into the clathrin-independent membrane bending and dynamin-independent endocytic scission processes.
The NCBS-IC post-doctoral fellow will formulate a project in close collaboration with the two laboratories, and work at both sites during the duration of the fellowship, depending on the demands of the science.
Thottacherry, J. J. et al. (2018) Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells. Nat. Commun. 9, 4217.
Sathe, M. et al. (2018) Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis. Nat. Commun. 9, 1835.
Pezeshkian et al. (2017) Mechanism of Shiga toxin clustering on membranes. ACS Nano 11: 314-324
Shafaq-Zadah M, et.al. (2016) Persistent cell migration and adhesion rely on retrograde transport of beta1 integrin. Nat Cell Biol 18: 54-64
Renard H-F, et. al. (2015) Endophilin-A2 functions in membrane scission in clathrin-independent endocytosis. Nature 517: 493-496
Johannes L, Parton RG, Bassereau P, Mayor S (2015) Building endocytic pits without clathrin. Nat Rev Mol Cell Biol 16: 311-321
Lakshminarayan R. et.al (2014) Galectin-3 drives glycosphingolipid-dependent biogenesis of clathrin-independent carriers. Nat Cell Biol 16: 595-606
JOINT PROJECT NO. 2
Determining the mechanism of tubulin glycylation in stabilizing cilia and flagella
Microtubules are ubiquitous cytoskeletal elements with a variety of functions, the basic subunit, tubulin undergoes various posttranslational modifications (PTMs), which are involved in the determination of microtubule properties and functions. Among all known PTMs, glycylation is unique as it occurs only on axonemes, a highly specialized microtubule structures that build cilia and flagella. The Janke and Sirajuddin labs have previously demonstrated the importance of some tubulin PTMs in regulating motor activity, MAP binding and severing enzymes, however no molecular mechanisms has been identified for glycylation. So far we have shown that in vivo, glycylation is involved in the stabilization of cilia. To understand how glycylation can achieve this stabilization, we will now develop in vitro studies, which we will combine with cellular and in vivo approaches, to directly determine the molecular mechanism(s) by which glycylation stabilizes the microtubule axoneme in cilia and flagella. The in vitro approach will involve purifying tubulin/microtubules with defined glycylation patterns, as well as measuring the intraflagellar anterograde and retrograde motor activity, the stability of microtubules, and severing assays. Our two labs have established the complementary methodology to successfully perform the collaborative project. The Janke lab has established cell and mouse models for glycylation, and installed a pipeline for the production of à-la-carte tubulin for in vitro assays. The Sirajuddin team has a strong expertise in in vitro assays (motors, MAPs, severing enzymes), as well as in structural biology. The proposed project will complement our efforts in tackling the still enigmatic role of glycylation.
JOINT PROJECT NO. 3
We seek highly motivated, outstanding and adventurous candidates from the engineering and physical sciences for two (theoretical and experimental) NCBS-inStem-IC Postdoctoral Fellowships to investigate challenging problems in the Physics of Living Systems.
We propose to use the principles of soft condensed matter physics, fluid mechanics, and non-equilibrium physics to study the structure and form of complex compartments within the cell. Specifically, we are interested in the control of size, shape and spatial positioning of cellular organelles within the trafficking pathway, which are subject to active fission and fusion events. We view such cellular compartments as steady states of a driven non-equilibrium process, which we will investigate theoretically and experimentally.
Theory: We have recently constructed the covariant hydrodynamics of a closed membrane embedded in a Stokesian fluid which is subject to active fission and fusion events. These equations, solved analytically in simple settings, already reveal several exciting results, such as a generic drift instability of the organelle. To explore the full scope of these hydrodynamic equations, we would like to use numerical schemes, such as Boundary Element Methods (BEM) or Finite Element Methods (FEM). We are looking for adventurous and talented post-doctoral fellows who are familiar with or willing to learn these methods.
Experiment: We aim to build in vitro model lipid membrane systems incorporating membrane fusion, sorting and spontaneous scission using a combination of microfluidics and soft matter techniques. Using these basic ingredients, we will study the de novo emergence of features of complex organelles. Of particular emphasis will be the quantitative control of the transport, fusion, fission and segregation of the structures commensurate with in vitro studies. These experiments will be carried out in close collaboration with the theoretical investigations above. We are looking for expertise in artificial membrane systems, microfluidics, microscopy, optical manipulation and related physical techniques. Persons with a theoretical background with a strong interest in building experiments are also encouraged to apply.
This is a joint research program between theorists (Prof. Madan Rao and Prof. Pierre Sens) and experimentalists (Prof. Patricia Bassereau and Dr. Shashi Thutupalli) at two institutes (National Centre for Biological Sciences, Bangalore, India and the Institut Curie, Paris, France).
Right from the start, the candidate will be encouraged and even expected to develop a great degree of independence — from drafting the initial details of the project within the broad framework outlined and to propose interesting directions going forward.
Please send your application (CV, a list of publications and two reference letters) by email to firstname.lastname@example.org, email@example.com, firstname.lastname@example.org, email@example.com before 1 March 2019.