Phosphatidic Acid Dependent Recruitment of Microtubule Motors to Spherical Supported Lipid Bilayers for In-vitro Motility Assays
Pankaj Kumar, Paulomi Sanghavi, Dwiteeya Chaudhury, Roop Mallik . Accepted (Cell Reports)
Older version of Manuscript :-BioRXiv (2023)

In Vivo Trapping of Latex Bead Phagosomes for Quantitative Force Measurements.
Paulomi Sanghavi, Arpan Rai, Roop Mallik. Methods in Molecular Biology 2623:187-200 (2023). 

Microtubule Motor Driven Interactions of Lipid Droplets: Specificities and Opportunities (Review article)
Jagjeet Singh, Paulomi Sanghavi and Roop Mallik. Frontiers in Cell and Developmental Biology, September 2022. 

Metabolic and Immune Sensitive Contacts between Lipid droplets and Endoplasmic Reticulum Reconstituted In vitro
Sukrut Kamerkar, Jagjeet Singh, Subham Tripathy, Hemangi Bhonsle, Mukesh Kumar, Roop Mallik.   PNAS Vol. 119, No. 24 (2022)              
News Article at Research Matters                  Older version :-  BioRxiv June 7th 2021.
The individual compartments inside a living cell are factories that must constantly exchange material (e.g. lipids and proteins) for the Cell to function. Such exchanges happen at Membrane Contact Sites (MCS) that form across sub-cellular compartments such as the Endoplasmic reticulum (ER), Lysosomes, Mitochondria, Lipid droplets etc. However, these MCS are notoriously difficult to study inside cells because of their complex and crowded geometry. Here we purify vesicles enriched in the ER from the Liver of a rat, and deposit these vesicles on a coverslip to create a lipid/protein containing membrane that mimics the ER. We then form MCS in a simplified planar geometry between this ER-mimic and Lipid droplets purified from rat liver. An Optical trap is used to demonstrate physical tethering at MCS, which changes dramatically in response to metabolic state and immune activation. This simplified assay could possibly be used in future to screen for drugs that can regulate Lipid droplet – ER contacts in the liver. 

Mapping Sphingolipid Metabolism Pathways during Phagosomal Maturation.
Neelay Mehendale, Roop Mallik and Siddhesh S. Kamat.  ACS Chem. Biol. 16, 2757 (2021)
We had shown that ceramides are enriched on Late Phagosomes, and this requires activity of Ceramide synthase 2 (CerS2). Surprisingly, CerS2 activity was same in Early Phagosomes (EPs) and Late Phagosomes (LPs). Then, where does the Ceramide made by CerS2 on EPs disappear? Here we find that a Neutral Ceramidase is active on Phagosomes. The Neutral Ceramidase converts Ceramide to Sphingosine on EPs. However, it gets inactivated on the LP because the pH is now acidic, and therefore Ceramide starts accumulating on the LP. Concomitant to increased ceramides, glucosylceramides are also substantially elevated on LPs. This finding has implication for Gaucher’s disease that is caused by accumulation of glucosylceramide. Work done in collaboration with S. Kamat’s group, IISER Pune.

ON and OFF Controls within Dynein-Dynactin on Native Cargoes.
Paulomi Sanghavi, Pankaj Kumar, Ankit Roy, M.S.Madhusudhan and Roop Mallik.   PNAS Vol 118 No. 23 (2021)
Dynein-dynactin is a Nanoscale Motor that generates force to drive vastly diverse cellular processes. We show that the dynein-dynactin linkage elevates Kon, so that the motor is activated and can generate force rapidly. In contrast, the dynactin-microtubule linkage has a completely different role – it lowers Koff, i.e. it prevents dynein’s detachment from microtubules. Our findings call attention to a less studied property of dynein–dynactin, namely its detachment against load, to understand dynein dysfunctions that cause neurological disorder.

Lis1 co-localizes with actin in the Phagocytic cup and regulates Phagocytosis. 
Aditya Chhatre, Paulomi Sanghavi and Roop Mallik.   Cytoskeleton (Wiley) 2020,77:249–260.       Highlighted on PreLights.  
Lissencephaly-1 (Lis1), a regulator of Dynein is found in the phagocytic cup. An optimum level of Lis1 appears necessary for particle engulfment. The studies are replicated in mouse macrophages and  Dictyostelium, bringing out an evolutionarily conserved role for Lis1 in phagocytosis.ole for Lis1 in phagocytosis.

From Physics to Physiology at the Membrane-Motor Interface (Comment).
Roop Mallik,   Nature Reviews Molecular Cell Biology 2020, 21:61–62. 
Force-generating motor proteins usually assemble on lipid membranes inside cells. We draw attention to the membrane as a dynamic entity that responds to cellular and metabolic demands, thereby harnessing the force from motors to control pathogen degradation, cancer metastasis, lipid homeostasis and possibly other functions relevant to health and physiology.

Insulin activates Intracellular transport of Lipid Droplets to release Triglycerides from the Liver.
Mukesh Kumar, Srikant Ojha, Priyanka Rai, Alaumy Joshi, Siddhesh S. Kamat and Roop Mallik.  The Journal of Cell Biology, 218 (11) 3697-3713 (2019)
Recommended at Faculty of 1000 prime           
We describe a pathway for channelling fat from the liver into blood in a controlled manner across fed/fasted cycles. Insulin, phosphatidic acid and kinesin collaborate inside hepatocytes to deliver fat-containing lipid droplets at the smooth-ER, which are then catabolised to supply fat for lipoprotein production and secretion. Further, we leverage this knowledge to show that a peptide can block fat secretion from liver cells, thus possibly targeting hyperlipidemia.

Cytoskeletal Motors:Structure and Function in Hepatocytes (Book Chapter)
Mukesh Kumar, Arnab Gupta and Roop Mallik. The Liver : Biology and Pathobiology, 6th Edition

Dynein in Endosome and Phagosome Maturation (Book Chapter)
Ashim Rai, Divya Pathak & Roop Mallik. The Handbook of Dynein, 2nd Edition 

Lipidomics suggests a new role for Ceramide Synthase in Phagocytosis
Divya Pathak, Neelay Mehendale, Shubham Singh, Roop Mallik, and Siddhesh S Kamat.  ACS Chemical Biology, 13, 2280-2287 (2018)
We show that membrane-associated ceramide increases by more than two-fold on phagosomes as they mature inside macrophage cells. The enzyme ceramide synthase appears responsible for this increase. We speculate that ceramide helps assemble stable and mechanically rigid lipid-platforms, where motor proteins assemble to generate force and transport the phagosome. Work done in collaboration with S. Kamat’s group, IISER Pune.

Fluorescence microscopy applied to intracellular transport by microtubule motors  (Review)
Divya Pathak, Shreyasi Thakur and Roop Mallik.  Journal of Bioscience, 43, 437–445 (2018)
Fluorescence microscopy has been used to address a variety of questions related to Microtubule Motor driven Intracellular transport such as Motor–organelle selectivity, Structural determinants of processivity, Collective behaviour of motors, Track selection etc. We review efforts to understand these sub-microscopic machines using fluorescence based approaches.

Coin-tossing explains the Activity of Opposing Microtubule Motors on Phagosomes.
Paulomi Sanghavi, Ashwin D’Souza, Ashim Rai, Arpan Rai, Ranjith Padinhatheeri and Roop Mallik.  Current Biology 28, 1460–1466 (2018)
We analyse optical trapping data on a cellular organelle that is being carried around in the form of a “cargo” by dynein and kinesin. It is suggested that a hypothetical coin is being repeatedly tossed to decide which motor (dynein or kinesin) will be activated on a cargo. In other words, the activity of motors is stochastic, independent of each other, and can be described as a process with no memory — a Markov chain. The “Coin-tossing model” provides a conceptual framework for understanding the distribution of organelles inside cells resulting from their bidirectional transport on microtubules. A single parameter, i.e. the fairness of this coin, could decide the spatial organization of a variety of components within cells.

Kinesin dependent Mechanism for Controlling Triglyceride Secretion from the Liver.
Priyanka Rai, Mukesh Kumar, Geetika Sharma, Pradeep Barak, Saumitra Das, Siddhesh S. Kamat and Roop Mallik.   PNAS 114, 12958–12963 (2017).
Commentary on this article in  HEPATOLOGY   Article in the BIOCHEMIST   WHO LET THE FAT OUT.   
Elevated Serum Triglyceride is bad news when it shows up in a blood report. This is because excess triglyceride (fat) circulating around in blood is the beginning of obesity, diabetes, heart disease – in general, all that is not good. To keep you healthy, your liver must secrete triglyceride/fat in the form of VLDL into blood in precisely controlled manner. But, how is this secretion controlled inside the liver at the level of molecules and cells? We show that the motor protein Kinesin transports fat-containing particles (called lipid droplets) to locations inside liver cells from where the fat is secreted out. Most interestingly, because this transport is controlled by insulin, it increases in Fed state but decreases in Fasted state of the animal. Kinesin therefore allows the liver to control how much of it’s internally stored fat is secreted out, and this in turn maintains constant circulating fat in our body. We also find that the same mechanism may allow hepatitis-C virus (HCV) to replicate inside liver cells. Collaboration with S. Kamat’s group and S. Das group.

Feeding-Fasting dependent recruitment of Membrane Microdomain proteins to Lipid Droplets purified from the Liver.
Kritika Sadh, Priyanka Rai and Roop Mallik.   PLoS ONE 12(8): e0183022 (2017).
We show that two proteins (Flotillin-1 and SNAP23) that bind to membrane microdomains associate differently with lipid droplets purified from rat liver depending on the feeding/fasting state of the animal. Our work brings out physiologically relevant aspects of lipid droplet biology that are different from, and may not be entirely possible to replicate and study in cell culture.

Isolation of Latex Bead Phagosomes from Dictyostelium for in vitro Functional Assays  (Protocol).
Ashwin D’Souza, Paulomi Sanghavi, Ashim Rai, Divya Pathak and Roop Mallik.   Bio-protocol 6(23): e2056 (2016).
A protocol to purify latex bead phagosomes (LBPs) from Dictyostelium. Purified LBPs can be used for in vitro motility and other functional assays in which several aspects of their behaviour inside cells is replicated.

Lipid – Motor Interactions : Soap Opera or Symphony?   (Review).
Divya Pathak and Roop Mallik.   Current opinion in Cell Biology (2016).
We review how lipids may orchestrate the recruitment of motor proteins to a membrane. We also discuss how heterogeneity and local mechanical properties of the membrane may influence function of motor-teams. These issues gain importance because phagocytosed pathogens use lipid-centric strategies to manipulate motors and survive in host cells.

Inositol hexakisphosphate kinase 1 (IP6K1) activity is required for cytoplasmic dynein-driven transport.
Chanduri M, Rai A, Malla AB, Wu M, Fiedler D, Mallik R, Bhandari R.   Biochemical Journal  473(19):3031-47 (2016).
This study provides evidence for the involvement of IP6Ks in dynein function and proposes that inositol pyrophosphate-mediated pyrophosphorylation may act as a regulatory signal to enhance dynein-driven transport.

Dynein clusters into Lipid Microdomains on Phagosomes to drive Rapid Transport towards Lysosomes.
Ashim Rai, Divya Pathak, Shreyasi Thakur, Shampa Singh, Alok Kumar Dubey and Roop Mallik.   Cell 164, 722–734 (2016)
SPOTLIGHT on this article,    Nirschl et al Trends in Cell Biology (2016)      Article on CuriousCascade.
We use optical trapping to measure the force exerted when Motors carry phagosomes towards lysosomes, so that the bacteria enclosed within phagosomes can be killed. The lipid membrane covering a phagosome gets enriched in cholesterol as the phagosome matures, thus generating cholesterol-rich platforms (lipid rafts) on which many dynein motors assemble as a team. Once such a team is assembled, dyneins can generate large collective force to transport the phagosome (and enclosed bacteria) to its death. Therefore, Cholesterol, that much-hated molecule may also be needed to keep you safe from infections !!

Reconstitution of microtubule-dependent organelle transport     (Protocol).
Pradeep Barak, Ashim Rai, Alok Kumar Dubey, Priyanka Rai and Roop Mallik.   Methods in Enzymology 540, 231–248 (2014).
We describe protocols developed by us for in vitro motility of phagosomes and lipid droplets along microtubules. Such assays address basic questions about phagosome maturation and  fatty acid regulation. The in vitro assay allows controlled intervention to address mechanistic details, but is still physiologically relevant because endogenous organelles are being used. These protocols could also serve as a starting point to reconstitute the motility of other kinds of organelles.

Teamwork in Microtubule Motors (Review).
R. Mallik, Arpan K. Rai, Pradeep Barak, Ashim Rai and Ambarish Kunwar.   Trends in Cell Biology 23, 575-582 (2013).
What makes a motor work well in a team ? We review how collective force-generating properties of microtubule motors emerge from their single-motor properties.

Molecular adaptations allow Dynein to generate large collective forces inside cells.
Arpan K. Rai, Ashim Rai, Avin J. Ramaiya, Rupam Jha and Roop Mallik.   Cell 152, 172–182 (2013)  
Commentary in Developmental Cell    Editor’s Choice  in Science       Faculty of 1000 prime            Article  in “The Scientist“.
Biological processes often require large forces that can only be generated by many motor proteins working as a team. Intriguingly, large persistent force in many cellular processes is generated by weak and inefficient Dynein motors. Why is this counter-intuitive choice made?  To investigate, we directly measure using optical tweezers the force exerted by motor-teams on single phagosomes inside living cells. We demonstrate that N-dynein collective function improves linearly with N, whereas this is not true for kinesin. Thus, appropriately sized Dynein-teams can be assembled to generate force tailored to diverse cellular requirements. We find that Dynein slows down rapidly against opposing force, while proportionately increasing the force it generates itself (as if it has an automatic gear). Further, when pulled back with strong force, Dynein engages a catch-bond  to remain attached tenaciously to the microtubule. We describe a mechanism by which these molecular properties allow many Dyneins to share load equally, and therefore work efficiently as a team. Our findings trace the collective function of motors inside cells down to their single-molecule nanomechanical properties.

Quantitative optical trapping on single organelles in cell extract (New Technique Development).
Pradeep Barak, Ashim Rai, Priyanka Rai and Roop Mallik.   Nature Methods 10, 68–70 (2013).
We develop a simple optical trapping method to measure the force generated by motor proteins on a cellular organelle of unknown size. This overcomes the limitation of using artificial motor-coated beads of known size for optical trapping. This method now permits the more biologically relevant functional interrogation of native-like motor complexes resident on real organelles. We use this method to measure the force, number and activity of kinesin on motile lipid droplets isolated from the liver of normally fed and food-deprived rats. A marked reduction in kinesin activity is seen for the food-deprived rats.

Tug-of-war between Dissimilar Teams of Microtubule Motors Regulates Transport and Fission of Endosomes.
V Soppina, A K Rai, A J Ramaiya, P Barak and Roop Mallik.   PNAS 106, 19381 (2009).
How motor proteins effect endosome transport and fission is investigated through quantitative biophysical measurements. Opposing teams of Kinesin and Dynein are shown to engage in a molecular tug-of-war on single endosomes. Surprisingly, many (4 to 8) weak and detachment-prone dyneins are in tug-of-war against a single strong and tenacious kinesin. We show how this clever choice of opposing motor-teams allows efficient minus-end transport (required for endosome maturation) concurrently with controlled fission of endosomes (required for receptor recycling). This appears to be the first demonstration that the two major microtubule motors (Kinesin and Dynein) function differently at the molecular level inside cells, and of how this difference may regulate an important cellular process, namely endosome biogenesis.

Simple non-fluorescent polarity labeling of Microtubules for Molecular Motor assays (New Technique Development).
V Soppina, A K Rai and Roop Mallik.   Biotechniques 46, 297 (2009).
A new method to identify the polarity (minus and plus) of in vitro assembled microtubules. We reconstitute motion of endosomes extracted from cells on the labelled microtubules. The opposing activity of dynein (moves towards minus-end of MT) and kinesin (moves towards plus-end) on individual endosomes can be resolved with high spatial and temporal resolution. This will help understand how these two opposing motors compete while moving their cargo (e.g. the endosome) on a microtubule inside cells.

Postdoctoral (2001 – 2005)

Studying Molecular Motor-based Cargo Transport: What is Real, and What is Noise. 
D. Petrov, R. Mallik, G.T. Shubeita, M. Vershinin, S.P. Gross, and C. Yu, Biophysical Journal 92, 2953 (2007)

Building complexity: an in vitro study of cytoplasmic dynein with in vivo implications.
R. Mallik,D. Petrov, S. A. Lex, S.J. King and S.P. Gross, Current Biology 15, 2075 (2005)
Commentary on this paper in Current Biology

Monte Carlo modeling of Single Molecule Cytoplasmic Dynein.
M.P. Singh, R. Mallik, S.P. Gross and C. Yu, Proceedings of the National Academy of Sciences, USA (PNAS) 102, 12059 (2005).  

Molecular motors: Strategies to get along. (Review)
R. Mallik and S.P. Gross, Current Biology 14, R971 (2004)

Cytoplasmic Dynein functions as a gear in response to load.
R.Mallik, B.C. Carter, S.A. Lex, S.J. King and S.P. Gross, Nature 427, 649-652 (2004)
Commentary on this paper in Current Biology 

Kinetics of proton transfer in a green fluorescent protein: A laser-induced pH jump study.
R. Mallik, J.B. Udgaonkar and G. Krishnamoorthy, Proceedings of Indian Academy of Sciences (Chem Sci) 115, 307-317 (2003) 

Ph.D. – Condensed Matter Physics (1994 – 2000)
  1. E.V. Sampathkumaran, R. Mallik, P.L. Paulose & S. Majumdar (2000). Silence of magnetic layers to magnetoresistive process and eletronic separation at low temperatures in (La,Sm)Mn2Ge2 Phys. Lett. 71, 123.
  2. E.V. Sampathkumaran, Subham Majumdar, R. Mallik, R.Vijayraghavan, H. Wada and M. Shiga (2000). Unusually large negative magnetoresistance around 200 K in the alloys Gd1-xLax Mn2Ge2 J. Phys.: Cond. Matter 12, L399. 
  3. R. Saha, H. Sugawara, T. D. Matsuda, H. Sato, R. Mallik and E. V. Sampathkumaran (1999). Magnetic anisotropy, first-order-like metamagnetic transitions and large negative magnetoresistance in the single crystal of Gd2PdSi3. Phys. Rev. B 60, 12162.
  4. S. Majumdar, R. Mallik, E.V. Sampathkumaran, K. Rupprecht and G. Wortmann (1999) Magnetic behaviour of Eu2CuSi3 : Large negative magnetoresistance above Curie temperature Phys. Rev. B 60, 6770.
  5. Subham Majumdar, M. Mahesh Kumar, R. Mallik and E. V. Sampathkumaran (1999). La substitution induced linear temperature dependence of electrical resistivity and Kondo behaviour in the alloys, Ce2-xLaxCoSi3 Solid State Communications 110, 509. 
  6. R. Mallik, E.S. Reddy, P.L. Paulose, S. Majumdar and E.V. Sampathkumaran(1999). Transport and magnetic anomalies due to A-site ionic-size mismatch in La0.5Ca0.5-xBax MnO3 . J. Phys.: Cond. Matter 11, 4179.
  7. S. Mazumdar, R. Mallik, E.V. Sampathkumaran, P.L. Paulose and K. V. Gopalakrishnan(1999). Unexpected modication of magnetic properties by Y substitution in Eu2PdSi3. Phys. Rev. B 59, 4244.
  8. R. Mallik, E.V. Sampathkumaran, P.L. Paulose, H. Sugawara and H. Sato(1998). Magnetic anomalies in Gd2PdSi3. Pramana – J. Phys. 51, 505.
  9. S. Mazumdar, R. Mallik, E.V. Sampathkumaran and P.L. Paulose(1998). Residual resistivity ratio and its relation to the magnetoresistance in LaMn2Ge2 derived alloys. Solid State Communications 108, 349.
  10. R. Mallik and E.V. Sampathkumaran(1998) Magnetic precursor effects, electrical and magnetoresistance anomalies, and heat capacity behaviour of Gd alloys. Phys. Rev. B 58, 9178.
  11. R. Mallik, E.V. Sampathkumaran, J. A. Alonso and M. J. Martinez- Lope(1998). Complex low temperature transport behavior of RNiO3 – type of compounds. J. Phys.: Cond. Matter 10, 3969.
  12. R. Mallik, E.V. Sampathkumaran, P.L. Paulose, M. Strecker, G. Wortmann and Y. Ueda(1998). Complex magnetism in a new alloy, Eu2PdSi3, with two crystallographically inequivalent sites. J. Magn. Magn. Mater. 185, L135 (Letters).
  13. R. Mallik, E.V. Sampathkumaran and P.L. Paulose(1998). Large low temperature magnetoresistance and magnetic anomalies in Tb2PdSi3 and Dy2PdSi3. Solid State communications 106, 169.
  14. R. Mallik, E.V. Sampathkumaran, M. Strecker and G. Wortmann(1998). Observation of a minimum in the temperature dependent electrical resistance above magnetic ordering in a Gd alloy. Europhysics Letters 41, 315.
  15. A. Sundaresan, P. L. Paulose, R. Mallik and E. V. Sampathkumaran (1997). Bandwidth controlled magnetic and electronic transitions in La0.5Ca0.5-xSrxMnO3 distorted perovskite. Phys. Rev. B 57, 2694.
  16. R. Mallik, E.V. Sampathkumaran and P.L. Paulose(1997). Large positive magnetoresistance at low temperatures in a ferromagnetic natural multilayer, LaMn2Ge2 Applied Physics Letters 71, 2385.
  17. R. Mallik, P.L. Paulose, E.V. Sampathkumaran, S. Patil and V. Nagarajan (1997). Coexistence of localized and (induced) itinerant magnetism and heat-capacity anomalies in Gd1-xYx Ni alloys Phys. Rev. B 55, 8369.
  18. R. Mallik, E.V. Sampathkumaran, P.L. Paulose and V. Nagarajan (1997) Magnetoresistance in GdNi: Magnetic polaronic-like effect near the Curie temperature and low-temperature sign reversal Phys. Rev. B 55, R8650 (Rapid Communication).
  19. R. Mallik, E.V. Sampathkumaran and P.L. Paulose (1997). Kondo lattice behaviour and multiple characteristic temperatures in CeIr2 Ge2 Phys. Rev. B 55, 3627.
  20. R. Mallik, E.V. Sampathkumaran, J. Dumschat and G. Wortmann (1997). Magnetic ordering and spin fluctuation behaviour in compounds of the type Ce2(Pd,Rh)2In. Solid State Communications 102, 59.
  21. R. Mallik and E.V. Sampathkumaran (1996). Magnetic behaviour of the alloys (Ce1-x Yx)2PdSi3 J. Magn. Magn. Mater. 164, L13.
  22. E.V. Sampathkumaran, P.L. Paulose and R. Mallik (1996). Magnetoresistance anomalies and multiple magnetic transitions in SmMn2Ge2. Phys. Rev. B 54, R3710 (Rapid Communication).
Comments / Conference proceedings etc.
  1. R. Mallik and Steven P. Gross Molecular motors as cargo transporters in the cell The good, the bad and the ugly  Physica A 372, 65 (2006). Proceedings of the Workshop on “Common Trends in Traffic Systems”, IIT-Kanpur, Feb 2006 Download PDF
  2. R. Mallik and Steven P. Gross  Intracellular Transport: How Do Motors Work Together? Current Biology 19 : R416 (2009)
  3. A.M. Saxena, R. Mallik, J.B. Udgaonkar and G. Krishnamoorthy, Protein dynamics controls proton transfer from bulk solvent to interior of proteins: a case study with GFP. 8th Conference on Methods and Applications of Fluorescence: Spectroscopy, Imaging and Probes. Prague, (2003)
  4. A.M. Saxena, R. Mallik, J.B. Udgaonkar and G. Krishnamoorthy. Protein Dynamics Controls the Fast Kinetics of Proton Transfer from Bulk solvent to Interior of Proteins: A case Study with GFP. Symposium of the Protein Society: Protein Structure & Function, Indian Institute of Technology Bombay, Mumbai, India (2003)
  5. Majumdar et. al. Physica B 281&282 (2000) 367 Majumdar et. al. Physica B 259&261 (1999) 166
  6. R. Mallik et. al. Physica B 259&261 (1999) 892 Majumdar et. al. Physica B 259&261 (1999) 843
  7. R. Mallik et. al. Physica B 230&232 (1997) 169 Mallik et. al. Physica B 230&232 (1997) 731
  8. Sampathkumaran et. al. Physica B 223&224 (1996) 316
  9. R. Mallik et. al. Physica B 223&224 (1996) 382