Manoj Gopalakrishnan

Abstract: We study the velocity-force (V-F) relation for a Brownian ratchet consisting of a linear rigid polymer growing against a diffusing barrier, acted upon by a opposing constant force (F). Using a careful mathematical analysis, we derive the V-F relations in the extreme limits of fast and slow barrier diffusion. In the first case, V depends exponentially on the load F, in agreement with the well-known formula proposed by Peskin, Odell and Oster (1993), while the relationship becomes linear in the second case. For a bundle of two filaments growing against a common barrier, equal sharing of load in the corresponding V-F relation is predicted by a mean-field argument in both limits. However, the scaling behaviour of velocity with the number of filaments is different for the two cases. In the limit of large D, the validity of the mean-field approach is tested, and partially supported by a detailed and rigorous analysis. Our principal predictions are also verified in numerical simulations. Graphic abstract: [Figure not available: see fulltext.].

Normal thermal fluctuations of the cell membrane have been studied extensively using high resolution microscopy and focused light, particularly at the peripheral regions of a cell. We use a single probe particle attached non-specifically to the cell-membrane to determine that the power spectral density is proportional to (frequency)-5/3 in the range of 5 Hz to 1 kHz. We also use a new technique to simultaneously ascertain the slope fluctuations of the membrane by relying upon the determination of pitch motion of the birefringent probe particle trapped in linearly polarized optical tweezers. In the process, we also develop the technique to identify pitch rotation to a high resolution using optical tweezers. We find that the power spectrum of slope fluctuations is proportional to (frequency)-1, which we also explain theoretically. We find that we can extract parameters like bending rigidity directly from the coefficient of the power spectrum particularly at high frequencies, instead of being convoluted with other parameters, thereby improving the accuracy of estimation. We anticipate this technique for determination of the pitch angle in spherical particles to high resolution as a starting point for many interesting studies using the optical tweezers. This journal is

When transporting cellular cargoes along the cytoskeletal filament track, motor proteins produce additional frictional drag. This "protein friction"determines the mean speed of a cargo for a given force and the energy dissipated per chemical cycle. Motor protein friction has been measured directly in an optical tweezer experiment, and can also be estimated from the force-velocity curve, close to stall. We present a mathematical and computational study of this phenomenon. In our model, a motor protein is elastically linked to a μm-sized cargo particle, and undergoes tightly coupled, biased random walk-like motion on the microtubule filament, the bias being contributed by nucleotide hydrolysis as well as stretching of the linker "spring", with spring constant κ. The cytoplasm is assumed to be a Newtonian fluid, which exerts a damping force γ V on the cargo moving with velocity V. The effective drag coefficient γeff = F/V is measured in our numerical simulations, where F is the net external force on the cargo, including motor-induced force, near F = 0. The motor friction γm = γeff- γ is predicted theoretically and compared with simulation data for a range of values of κ and γ. The predicted values for small γ are found to be similar to experimental results, though smaller in magnitude. Numerical simulations also show that γm is a weakly increasing function of γ, and is additive when multiple motors are involved in transportation.

We study the normal fluctuations of an MCF-7 cell membrane and calibrate the optical trap to detect pitch motion to get information about the rocking motion of a birefringent particle. We could show both theoretically and experimentally that the Z power spectrum has a power-law behavior of (frequency)-5/3, and We find that the power spectrum of slope fluctuations is proportional to (frequency)-1. We could extract parameters like bending rigidity directly from the power spectrum fitting parameters in 5 Hz to 1 kHz range. Our method was powerful enough to identify pitch rotation for a spherical birefringent particle to a high resolution using optical tweezers.

In the intracellular environment, the intrinsic dynamics of microtubule filaments is often hindered by the presence of barriers of various kind, such as kinetochore complexes and cell cortex, which impact their polymerisation force and dynamical properties such as catastrophe frequency. We present a theoretical study of the effect of a forced barrier, also subjected to thermal noise, on the statistics of catastrophe events in a single microtubule as well as a 'bundle' of two parallel microtubules. For microtubule dynamics, which includes growth, detachment, hydrolysis and the consequent dynamic instability, we employ a one-dimensional discrete stochastic model. The dynamics of the barrier is captured by over-damped Langevin equation, while its interaction with a growing filament is assumed to be hard-core repulsion. A unified treatment of the continuum dynamics of the barrier and the discrete dynamics of the filament is realized using a hybrid Fokker-Planck equation. An explicit mathematical formula for the force-dependent catastrophe frequency of a single microtubule is obtained by solving the above equation, under some assumptions. The prediction agrees well with results of numerical simulations in the appropriate parameter regime. More general situations are studied via numerical simulations. To investigate the extent of 'load-sharing' in a microtubule bundle, and its impact on the frequency of catastrophes, the dynamics of a two-filament bundle is also studied. Here, two parallel, non-interacting microtubules interact with a common, forced barrier. The equations for the two-filament model, when solved using a mean-field assumption, predicts equal sharing of load between the filaments. However, numerical results indicate the existence of a wide spectrum of load-sharing behaviour, which is characterized using a dimensionless parameter.

We explore correlations between dynamics of different microtubules in a bundle, via numerical simulations, using a one-dimensional stochastic model of a microtubule. The guanosine triphosphate (GTP)-bound tubulins undergo diffusion-limited binding to the tip. Random hydrolysis events take place along the microtubule and converts the bound GTP in tubulin to guanosine diphosphate (GDP). The microtubule starts depolymerising when the monomer at the tip becomes GDP-bound; in this case, detachment of GDP-tubulin ensues and continues until either GTP-bound tubulin is exposed or complete depolymerisation is achieved. In the latter case, the microtubule is defined to have undergone a “catastrophe”. Our results show that, in general, the dynamics of growth and catastrophe in different microtubules are coupled to each other; the closer the microtubules are, the stronger the coupling. In particular, all microtubules grow slower, on average, when brought closer together. The reduction in growth velocity also leads to more frequent catastrophes. More dramatically, catastrophe events in the different microtubules forming a bundle are found to be correlated; a catastrophe event in one microtubule is more likely to be followed by a similar event in the same microtubule. This propensity of bunching disappears when the microtubules move farther apart.

We investigate temporal cooperative behaviour in a realistic model of a molecular motor-cargo assembly by simulating a one-dimensional stochastic model of a motor-bound bead in an optical trap. Here, each motor is linked to a bead through an elastic spring, and performs forward and backward hopping motions on the filament. The rates characterising the forward and backward steps are different because of the free energy change associated with ATP hydrolysis. Apart from forward and backward hopping, motor bind to, and unbind from the filament with specific rates, while remaining attached to the bead. The bead is subjected to elastic force originating from the stretched motor-springs, thermal noise as well as an external force from an optical trap. We constructed a Gillespie code which can simulate the dynamics of up to six motors. Our results indicate that the emergence of cooperative behaviour, as observed in experiments, is closely connected with the load-sharing properties of the team. Simulation results of the mean first passage time show excellent agreement with experimental data of the same in the literature.

We study the stationary states of an overdamped active Brownian particle (ABP) in a harmonic trap in two dimensions via mathematical calculations and numerical simulations. In addition to translational diffusion, the ABP self-propels with a certain velocity, whose magnitude is constant, but its direction is subject to Brownian rotation. In the limit where translational diffusion is negligible, the stationary distribution of the particle's position shows a transition between two different shapes, one with maximum and the other with minimum density at the center, as the trap stiffness is increased. We show that this nonintuitive behavior is captured by the relevant Fokker-Planck equation, which, under minimal assumptions, predicts a continuous phase transition-like change between the two different shapes. As the translational diffusion coefficient is increased, both these distributions converge into the equilibrium, Boltzmann form. Our simulations support the analytical predictions and also show that the probability distribution of the orientation angle of the self-propulsion velocity undergoes a transition from unimodal to bimodal forms in this limit. We also extend our simulations to a three-dimensional trap and find similar behavior.

Conventionally, only the normal cell membrane fluctuations have been studied and used to ascertain membrane properties like the bending rigidity. A new concept, the membrane local slope fluctuations was introduced recently (Vaippully et al 2020 Soft Matter 16 7606), which can be modelled as a gradient of the normal fluctuations. It has been found that the power spectral density (PSD) of slope fluctuations behave as (frequency)−1 while the normal fluctuations yields (frequency) − 5 / 3 even on the apical cell membrane in the high frequency region. In this manuscript, we explore a different situation where the cell is applied with the drug Latrunculin-B which inhibits actin polymerization and find the effect on membrane fluctuations. We find that even as the normal fluctuations show a power law (frequency) − 5 / 3 as is the case for a free membrane, the slope fluctuations PSD remains (frequency)−1, with exactly the same coefficient as the case when the drug was not applied. Moreover, while sometimes, when the normal fluctuations at high frequency yield a power law of (frequency) − 4 / 3 , the pitch PSD still yields (frequency)−1. Thus, this presents a convenient opportunity to study membrane parameters like bending rigidity as a function of time after application of the drug, while the membrane softens. We also investigate the active athermal fluctuations of the membrane appearing in the PSD at low frequencies and find active timescales of slower than 1 s.