ICTS

Quantitative Biology of the Cytoskeleton during Division, Differentiation and Disease

Biology is becoming increasingly more quantitative with the advent of a number of new techniques that make quantitation feasible. This advancement in the interpretation of biology has transformed it into aprecise science that can help comprehend life, health and disease down to the level of the numbers of molecules. One component of the cell that is central to all these states is the cytoskeleton. The cytoskeleton, as its name suggests, is the determinant of cell shape and form. Re-organization of the cytoskeleton and its associated proteins follows major cell fate decisions such as cell division, differentiation and disease. Although qualitative studies on some of the changes underlying these states of cells have been described using biochemistry, molecular biology and static visualization techniques, the vivid dynamics of the cytoskeleton and the associated proteins and their interplay during these processes have remained elusive. So too, an estimate of the numbers and the spatial organization of the players is lost when conventional biology techniques are applied to answer questions relating to the cytoskeleton. The talk will be focused on the application of quantitative techniques to investigate the regulation of the motor protein cytoplasmic dynein in vivo, the role of the microtubules in determining cell fate and in mitochondrial dynamics.
 

Mother-daughter information transfer through the coupling of replication fork movement and nucleosome dynamics

Positioning of nucleosomes along the genomic DNA is crucial for many cellular processes that include gene regulation and higher order packaging of chromatin. The question that how nucleosome positioning information from a parent chromatin gets transferred to the daughter chromatin is highly intriguing. Recent experiments have indicated that, during replication, the movement of the replication fork is strongly coupled to the nucleosome deposition behind it. In this talk, I will discuss one of our recent work that shows interplay between nucleosome removal timescale at the replication fork and nucleosome sliding behind the replication fork can give rise to an interesting phenomena where the daughter chromatin can inherit the precise nucleosome positioning of the parent, which is heterogeneous along the genome.
 

Investigating Mitochondrial Dynamics in Fission Yeast using Chemical Kinetics

The cytoskeleton drives many essential processes in vivo, but for this the system of filaments will arrange itself into different overall spatial organizations, e.g., random, branched networks, parallel bundles, antiparallel arrays, etc. A general objective of our research is to understand what makes these architectures adapted to their tasks. In this talk, I will first focus on 2D disorganized actin networks in which the filaments are oriented randomly in all directions, and are connected both by active molecular motors and passive crosslinkers. Systems with these properties have been reconstituted in vitro, and serve as a model of the cortical actomyosin networks that drive morphogenesis in animal tissues. Although the network components and their properties are known, the requirements for contractility are still poorly understood. I will describe a theory that predicts whether an isotropic network will contract, expand, or conserve its dimensions, depending on the properties of the filaments and the elements that connect them. The theory is simple and encompasses mechanisms of contractions previously proposed.
 

The 3.5Å cryo-EM structure of a fast dynein/dynactin complex

Dynein and its cofactor dynactin form a highly processive microtubule motor in the presence of an activating adaptor, such as BICD2. Different adaptors link dynein/dynactin to distinct cargos. Here we use electron microscopy (EM) and single molecule studies to show that adaptors can recruit a second dynein to dynactin. Whereas BICD2 is biased toward recruiting a single dynein, the adaptors BICDR1 and HOOK3 predominantly recruit two. We find that the shift toward a double dynein complex increases both force and speed. A 3.5 Å cryo-EM reconstruction of a dynein tail/dynactin/BICDR1 complex reveals how dynactin can act as a scaffold to coordinate two dyneins side by side. Our work provides a structural basis for how diverse adaptors recruit different numbers of dyneins and tune the motile properties of the dynein/dynactin transport machine.
 

Regulation and control of ribosome traffic on mRNA track: interface of physics and operations research in biology

Ribosome is a cyclic molecular machine that synthesizes proteins translating the corresponding genetic message. In each successful cycle, it steps forwards by one codon on the template mRNA track and elongates the nascent protein by a single amino acid. Signals encoded chemically on the sequence of the subunits of the mRNA and physically in its secondary structures are believed to control and regulate the nature and rate of protein synthesis. Moreover, often many ribosomes walk along the same mRNA strand, using it as the common track, while synthesizing distinct copies of the same protein. Such a poly-ribosome (polysome) resembles vehicular traffic on a highway. In recent years we have developed kinetic models of ribosome traffic that capture the key features under wide range of circumstances, particularly in unconventional translation. These models are developed by extending the Totally Asymmetric Simple Exclusion Process (TASEP), which is the simplest model of a system of interacting self-driven particles that can attain steady state far from equilibrium. The regulation and control of vehicular traffic has also been a traditional area of investigation in operations research. In this talk I'll present an overview of some results of our interdisciplinary research that lies at the interface of non-equilibrium statistical physics and operations research.
 

1. GREMAN, Université François Rabelais, Tours, France
2. Laboratoire de Physique des Solides, Université Paris-Sud, Orsay, France
3. Energy Science and Engineering, DGIST, Daegu, Korea
    

Insights into DNA – protamines self-assembly

Aggregation of strongly‐charged polymers (polyelectrolytes) has been extensively studied due to its importance in biological phenomena such as DNA packaging, gene regulation, and cytoskeleton organization. DNA has been one of the most studied strong polyelectrolyte and it has been shown that it can condense in vitro into various morphologies under a large variety of agents such as multivalent cations, poly-ions and basic proteins. Such effective attraction between like-charged polyelectrolytes is occurring for other semiflexible polyelectrolytes, indicating that specific interactions are not the main driving force. Instead, the short-range attraction has been theoretically ascribed to a certain form of positional correlations of the oppositely‐charged agents adsorbed along the polyelectrolyte chains.
Protamines are highly charged proteins compacting efficiently DNA during the spermatogenesis process. They are also used as non-toxic gene carriers for therapy purposes. Using salmon proteins (+21e) and short DNA fragments (146 bp), we have explored the physico-chemical conditions required for protamines to condense DNA. At low salt concentration, a set of experiments combining electrophoresis, light scattering and cryoelectron microscopy reveals the existence of small charged DNA–protamine complexes coexisting either with DNA or protamines in solution. We have used a coarse-grained model capturing the main molecular characteristics of DNAs and proteins and integrating out the atomistic degrees of freedom in order to probe larger timescale and length scale. Molecular dynamics and Langevin dynamics have been performed to explore the self-assembly phenomena. Aggregation is controlled solely by strong electrostatic interactions and spatial inhomogeneities introduced by the experimental protocol coupled to an order of magnitude difference in diffusion coefficients between DNA and proteins could play a crucial role in the self-assembly.

(1) Y. Lansac, J. Degrouard, M. Renouard, A.C. Toma, F. Livolant, E. Raspaud, Sci. Rep. 6, 21995 (2016)
(2) Raspaud E., Pelta J., de Frutos M., Livolant F., Phys. Rev. Lett. 2006, 97, 068103
(3) Toma A.C., de Frutos M., Livolant F., Raspaud E., Biomacromolecules, 2009, 10, 2129.
(4) Toma A.C., de Frutos M., Livolant F., Raspaud E., Soft Matter 2011, 7, 8847.
 

The Dynamics of the Ribosome as Revealed by Single-Particle Cryo-Electron Microscopy

Thanks to advances in image capture by direct electron detection cameras, single- particle cryo-electron microscopy (cryo-EM) has become a fast versatile technique of high-resolution structural biology. Cryo-EM, unlike X-ray crystallography, offers a way to visualize molecules in multiple conformations and binding states all equilibrating in the same sample. Thus, by suitable design of the experiment, the successive states of a processive molecular machine can be depicted all at once, revealing its dynamics in a gallery of snapshot. This exciting capability of cryo-EM, first realized by the introduction of maximum likelihood classification, is captured in the catch phrase “Story in a Sample.” The resolution of the individual reconstruction depends on the fraction of time the state is occupied, however, and imaging intermediate states at high spatial resolution may require a large overall data collection going into millions. To enrich the occupancy of such states, chemical interventions (antibiotics, nonhydrolyzable NTP analogs) or mutations are required.
These advances in single-particle cryo-EM have made it possible to depict ribosome dynamics during the stages of initiation, elongation, termination and recycling at a degree of detail inconceivable with other techniques. For example, tRNA selection, which involves discrimination between cognate and near-cognate codon-anticodon matches, has been dissected into a sequence of successive “microstates” showing the progress of base-pairing and ribosome engagement. Another example, mRNA-tRNA translocation is now understood as a complex interaction between the ribosome in a labile conformational state and EF-G. Two new developments are about to greatly expand the general toolset of single-particle cryo-EM. One is time-resolved cryo-EM using a mixing/spraying microfluidic chip, which enables the capture of short-lived intermediate states in a reaction. First results with this technique have been obtained for translation initiation, termination and ribosome recycling. The other development is in a more exhaustive analysis of the data collected in cryo-EM, doing full justice to conformational variability that is continuous, not discrete as current maximum likelihood methods assume. The ensemble method, developed in collaboration with Abbas Ourmazd and Peter Schwander at the University of Wisconsin at Milwaukee, employs manifold embedding of the cryo-EM snapshots and results in a mapping of the energy landscape of the molecule. Applied to the ribosome, new insights into the mechanism of translation are obtained.
 

Biophysical modeling to understand mitotic spindle assembly and stability

Mitotic spindles use an elegant bipolar architecture to segregate duplicated chromosomes with high fidelity. Bipolar spindles form from a monopolar initial condition in a fundamental construction problem. Microtubules, motors, and crosslinkers are important for bipolarity, but the mechanisms necessary and sufficient for spindle assembly remain unknown.

Computational modeling is a valuable tool to study biological self-assembly, because a small set of mechanisms can be modeled to determine whether they are sufficient to drive a complex process. We have developed a biophysical model that exhibits de novo bipolar spindle formation. We began with previously published data on fission-yeast spindle-pole-body size and microtubule number, kinesin-5 motors, kinesin-14 motors, and passive crosslinkers. Our model results agree quantitatively with our experiments in fission yeast, thereby establishing a minimal system with which to interrogate collective self-assembly.

By varying features of our model, we identify a set of functions essential for the generation and stability of spindle bipolarity. Kinesin-5 motors have puzzling motility properties that include slow speed, low processivity, and bidirectional motility or transport. We found that kinesin-5 bidirectional motility is essential for bipolar spindle assembly in our model. Another long-standing puzzle has been the observation that spindles form in cells lacking both kinesin-5 and kinesin-14 motors. We have studied how spindles can form in the presence of passive crosslinkers alone, and identify the requirements for this process. More recently, we have begun to investigate the mechanisms responsible for chromosome bi-orientation, correction of kinetochore-microtubule attachment errors, and lost kinetochore capture.

This talk will also briefly illustrate other current work on cytoskeletal active matter in our group.

Cooperative versus non-cooperative nature of a motor protein: New insights from an old theory

The microtubule-bound motors kinesin and dynein differ in many respects, a striking difference being that while kinesin is known to function mostly alone, dynein operates in large groups, much like myosin V in actin. In vitro experiments have shown that multiple kinesins tend to detach faster than single ones under stall, while larger assemblies of dyneins survive longer under stall. This makes dynein a team worker, capable of producing large collective force without detaching. In general, it is, however, unclear which biophysical properties of a single motor determine whether it is cooperative or non-cooperative. Within the limitations of a simplified picture of motor dynamics, we argue that this is determined by two dimensionless parameters: (i) the ratio of single molecule, load-independent detachment and attachment rates and (ii) the ratio of the applied force per motor to the detachment force of a single motor. We show that, in the large N limit, the (continuum) attachment-detachment dynamics of a N- motor assembly may be mapped to the motion of a Brownian particle in an effective potential. In this picture, cooperative behavior arises from the “trapping” of the particle in potential wells. For the latter problem, application of results from Kramers' theory predicts detachment times increasing exponentially with motor number, indicating cooperative behaviour. We show that a motor with load-insensitive detachment rate is always cooperative, whereas for a motor with detachment rate exponentially dependent on load, both cooperative and non-cooperative behaviour is possible. For the latter case, we also construct a "phase diagram" showing both regimes. The phase boundary predicted in our continuum theory agrees well with the exact results of Klumpp and Lipowsky (2005), and is consistent with the observed behaviour of kinesin and dynein.
 

Regulation of dynein-based transport

Microtubule minus-end transport is predominantly driven by groups of dynein motors, and a current topic of interest is how such transport is regulated. I will discuss our discovery of dynamic adaptation of force production, and after describing where such adaptation might be important, will discuss our new work investigating how such adaptation is controlled.
 

How the Nucleus of Human Cells is Structured by Entropy

Chromosomes due to dynamic association with protein factors fold to form three-dimensional structures. Many experiments have paved the way to understand folding and the nuclear architecture of the genome. Based on these experiments and models a fundamental understanding of the key principles that drive the physical organization of the genome in the nucleus has become possible through the concepts of loop and entropy. Using the concept of loop, models are now able to reproduce the results from several of the experiments within the framework of loop and entropy. Linking biological function to structure they moreover are able to make predictions and may help to carve out new ways treatment of diseases.
 

Traffic on filaments: Molecular motors under difficult conditions

Molecular motors power the busy traffic in cells, which is mostly takes place along the cytoskeletal networks of filaments, but also along DNA. This traffic is subject to perturbations and opposing forces of all kinds. Molecular crowding in the cytoplasm and other molecules bound to the track act as obstacles for movement including other motors moving on the same track. Other motors may try to pull the same cargo in a different direction, resulting in a tug-of-war. Finally, specific forces may attract cargoes, on such example are magnetosomes in magnetotactic bacteria, which attract each other magnetically, but need to be separated during cell division. In the talk I will discuss some recent theoretical work on these topics and illustrate how transport in cell deals with these perturbations.
 

Collective Dynamics Of Interacting Molecular Motors

Motor proteins, also known as biological molecular motors, are enzymatic molecules that convert chemical energy, typically obtained from ATP, into mechanical work and motion. They play important roles in functioning of biological systems by supporting cellular transport, cellular organization, transfer of genetic information and many other biologically relevant processes. Experiments indicate that many motor proteins interact locally, and molecular motors frequently function together in large groups. To understand the mechanisms of collective behavior of motor proteins we investigate the effect of interactions in the transport of molecular motors along linear filaments. Our analysis utilizes a recently introduced class of totally asymmetric exclusion processes that takes into account the intermolecular interactions via thermodynamically consistent approach. We develop a new theoretical method that allows us to compute analytically all dynamics properties of the system. Surprisingly, it is found that there is an optimal strength of interactions (weak repulsion) that leads to a maximal particle flux. It is also argued that correlations play important role in dynamics of interacting molecular motors. In addition, symmetry of interactions influences the dynamic properties of molecular motors. The biological implications of our findings are also discussed.
 

Tug-of-war between CDK1 and PP2A during mitosis regulates proper spindle orientation in human cells

TBA
 

Role of histone tails in NCP stability and chromatin assembly

TBA
 

The Dynein Catch Bond: Implications for cooperative transport

TBA
 

Cargo crowding at actin-rich regions along axons causes local traffic jams

Actin-rich regions in axons act as hotspots where moving cargo stall. Cargo crowding by stationary vesicles enhance stalling of any incoming cargo. Instead of permanent traffic jams, vesicles both stall and emerge from stationary vesicles in a motor and neuronal activity-dependent manner thus acting as dynamic reservoirs. Reversals preferentially occur at crowded regions along the axon. Our computational model suggests that such motion state changes of cargo at crowded regions is essential to maintain cargo flux.
 

1. Department of Biosciences and Bioengineering
2. Indian Institute of Technology Bombay Powai, Mumbai, India
    

Modeling in-vitro cargo switching at 3D intersections of microtubules

Eukaryotic cells employ specialized proteins called molecular motors for transporting organelles and vesicles from one location to another in a regulated and directed manner. These molecular motor proteins often work collectively as a team while transporting cargos which vary greatly in their shape and sizes-from spherical Lysosomes, Vacuoles and Peroxisomes to ellipsoid Mitochondria and Chloroplast, from almost cylindrical Golgi Bodies and small Microtubules to irregular shaped rRNA molecules and viral proteins. These cargos often navigate through complex three dimensional cytoskeletal network consisting of many microtubules and actin filaments to reach their destination, as single filaments are not long enough to traverse the entire dimensions of the cell from source to destination. Thus, cargos have to switch from one filament to another at these intersections during their intracellular travel for effective cell navigation. Here, we use mathematical and computational modeling to understand how cargo switching depends on geometries of microtubule intersections in 3D. 
 

Physical basis for self-assembly in living systems

In the talk we will discuss physical models to understand 3D organization and dynamics of bio-filaments, namely chromatin and microtubules. First, we will briefly discuss the importance of nucleosome positioning along DNA. Then we will present our work where we argue that DNA-bending non-histone proteins are important in understanding chromatin organization in 3D.

In the second part we will discuss shrinkage dynamics of microtubules. In the last part we will mention a physical model that can investi gate aggregation of proteins.
 

Regulation of microtubule assembly and stability by centrosomal proteins

The assembly dynamics of spindle microtubules plays an essential role in the accurate separation of the genetic materials. An interruption of the assembly and stability of spindle microtubules halts the progression of the mitotic cells and ultimately induces cell death. Several microtubule targeting compounds are used as anticancer drugs. These compounds either depolymerize microtubules or induce excessive polymerization of microtubules. Interestingly, the microtubule binders inhibit the dynamics of individual microtubules. Microtubule dynamics is regulated by several proteins in association with their posttranslational modifications. It has been shown that several centrosomal proteins influence the assembly and stability of microtubules. I will outline how centrosomal proteins regulate the assembly, disassembly and stability of microtubules.
 

A mechanistic model decodes MTOC clustering and nuclear migration in budding yeast during mitosis

Accurate chromosome segregation during mitosis is critical to maintain genome stability and prevent erroneous cell division. To this aim, a bipolar spindle will allow the sister chromatids to be pulled to opposite ends of the cell upon chromosomal separation, thus leading to the formation of two daughter cells with the correct chromosome number. While mammalian cells divide via open mitosis (nuclear envelop breaks down), chromosome segregation in budding yeast cells occur within the nucleus known as closed mitosis. In closed mitosis, the entire nucleus and the kinetochores cluster migrates into the daughter bud to facilitate high fidelity chromosome segregation. Further, kinetochore clustering and nuclear migration requires biased nucleation of microtubules. Our computer model, correctly predicts experimentally observed time-scales of nuclear migration and spindle assembly with error statistics. Further, we propose that in the absence of microtubule-bias, nucleus fails to move into the daughter bud, however, kinetochore clustering can still progress via redundant pathways.
 

Dynamics of Translation

Translation by the ribosome is a central and final step in gene expression in all organisms. The goal of our research is to understand the underlying conformational and compositional dynamics of translation. We have applied single-molecule fluorescence and structural approaches to both prokaryotic and eukaryotic translation systems. We have explored the underlying dynamics of translation initiation, elongation and termination. Here we will describe our work on translational elongation pausing caused by mRNA, nascent chains and antibiotic drugs. Our results show how small perturbations in translational components can lead to large impacts on protein synthesis.
 

When less is more - Logistics of Axonal Transport by Kinesin-2

Department of Biological Sciences, Tata Institute of Fundamental Research, Mumbai, India. Neurons and synapses constitute the logic flow network in a brain. Loss of the synaptic functions in the human brain is at the root of dementia and many other progressive neurodegenerative disorders such as Alzheimer's. Research around the globe has identified effects of several factors that can potentially cause such disorders. However, the critical trigger is still elusive. We studied this problem using Kinesin-2, a heterotrimeric motor protein comprising two different motor subunits and a non-motor accessory protein. It plays an essential role in the cilia/flagella assembly and maintenance, as well as in several other intracellular processes. We identified three independent intracellular transport systems involving kinesin-2 in axon: a soluble protein called Choline Acetyltransferase, the small GTPase Rab4, and a membrane-associated protein Acetylcholinesterase. These are also distributed in three distinct patterns in neurons. Experimental and theoretical analyses underlying these three transport processes revealed some interesting similarities, as well as highlighted the essential differences that could lead to the diversity in the transport modalities. Most importantly, we found a peculiar interdependent logistics that could maintain the synaptic balance in the neuronal network. I will discuss the results of our recent studies highlighting this conjecture.
 

Brownian motor at nano scale/molecular length scale

Brownian motors/Brownian ratchets are the microscopic version of motor which operate completely out of equilibrium. In this talk, I will dis cuss about the performance characteristics of a particular kind of Brownian ratchet model. Mainly I will talk about the effectiveness and coherence of transport governed by this kind of motor in different regime of parameter space of the model.
 

Selection of the initiator tRNA in the ribosomal P-site and regulation of ribosome heterogeneity in Escherichia coli

Initiator tRNAs (i-tRNAs) are special in possessing a highly conserved feature of the three consecutive GC base pairs (3GC pairs) in their a anticodon stems. In addition, the eubacterial i-tRNAs (tRNA fMet ) are characterized by the formylation of the amino acid attached to them. Translation initiation in eubacteria involves assembly of tRNA fMet , mRNA, initiation factors (IFs) and 30S ribosome in a 30S pre-initiation complex (30S pre-IC), which rearranges and joins 50S ribosome to form 70S IC. Upon releasing IFs, 70S IC becomes elongation-competent 70S. Using natural and engineered tRNAs, we show that while the property of formylation of tRNA fMet facilitates initial targeting of tRNA fMet to 30S ribosome, the 3GC pairs license tRNA fMet transitions from 30S to 70S IC, and then to elongation-competent 70S by release of IF3. Of the 3GC pairs, the middle GC pair (G30-C40), or merely G30 (in a specific context) suffices in this role, and is essential for the sustenance of E. coli. Participation of tRNA fMet in the first round of initiation complex formation licenses (via the 3GC pairs) the final steps of ribosome maturation by signaling RNases to trim the terminal extensions of immature 16S rRNA. Deficiency of tRNA fMet results in accumulation of ribosomes with immature 16S rRNA and translation of mRNAs with non-canonical Shine-Dalgarno sequences. Implication of the role of tRNA fMet , in generating proteome diversity will be discussed.
 

Controlling enzyme and polymerase activity on single DNA molecules

Optical tweezers allow us to probe the interactions of proteins with single DNA molecules and apply very small forces that alter these interactions. When protein binding or enzymatic activity change DNA length, these processes are altered by force. Here we investigate two processes that occur along single DNA molecule tracks in cells. In the first process, we probe the force-dependent polymerization and exonucleolysis of pol III core, the three-subunit subassembly of the E. coli replicative DNA polymerase III holoenzyme. By analyzing the force and concentration dependence of these two processes, we demonstrate that the process of switching between polymerase and exonuclease substrates is governed solely by primer stability, which changes with temperature, force, and the presence of mismatches. In a separate study, we also seek to understand how reverse transcriptase activity is regulated during HIV-1 replication. Here we probe the DNA binding interactions of human APOBEC3G, an innate antiviral immunity protein that functions as a cytidine deaminase enzyme. Our results show that APOBEC3G converts through dimerization from a fast enzyme that rapidly probes a single-stranded DNA track to a slow binding protein that is no longer an efficient enzyme. In addition, because of this slow binding, APOBEC3G dimers can act as a roadblock, likely preventing reverse transcription from occurring along the molecule, thereby inhibiting HIV-1 replication.