Biology subjects

Rajput, S. S.

Publications and source records attributed to Rajput, S. S..

5 recordsLinked to original sources

bioRxiv2023

Pathogenic aggregates alter actin organization and cellular viscosity resulting in stalled clathrin mediated endocytosis

Protein aggregation is a common underlying feature of neurodegenerative disorders. Cells expressing neurodegeneration-associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected due to altered actin cytoskeletal organization in the presence of Huntingtin aggregates. Additionally, we find that cells containing Huntingtin aggregates are stiffer and less viscous than their wild-type counterparts due to altered actin conformation, and not merely due to the physical presence of aggregate(s). We further demonstrate that CME and cellular viscosity can be rescued by overexpressing Hip1, Arp2/3 or transient LatrunculinA treatment. Examination of other pathogenic aggregates revealed that only a subset of these display defective CME, along with altered actin organization and increased stiffness. Together, our results point to an intimate connection between functional CME, actin organization and cellular stiffness in the context of neurodegeneration.

cell biology↗

bioRxiv2020

Viscoelasticity of single macromolecules using Atomic Force Microscopy

We measured viscoelasticity of single protein molecules using two types of Atomic Force Microscopes (AFM) which employ different detection schemes to measure the cantilever response. We used a commonly available deflection detection scheme in commercial AFMs which measures cantilever bending and a fibre-interferometer based home-built AFM which measures cantilever displacement. For both methods, the dissipation coefficient of a single macromolecule is immeasurably low. The upper bound on the dissipation coefficient is 5 x 10-7 kg/s whereas the entropic stiffness of single unfolded domains of protein measured using both methods is in the range of 10 mN/m. We show that in a conventional deflection detection measurement, the phase of bending signal can be a primary source of artefacts in the dissipation estimates. It is recognized that the measurement of cantilever displacement, which does not have phase lag due to hydrodynamics of the cantilever, is better suited for ensuring artefact-free measurement of viscoelasticty compared to the measurement of the cantilever bending. We confirmed that the dissipation coefficient in single macromolecules is below the detection limit of AFM by measuring dissipation in water layers confined between the tip and the substrate using similar experimental parameters. Further, we experimentally determined the limits in which the simple point-mass approximation of the cantilever works in off-resonance operation. Significance StatementSingle Macromolecules, including unfolded proteins bear rubber-like entropic elasticity and internal friction characterized by finite dissipation coefficient. Direct measurement of this viscoelastic response is important since it plays a significant role, both in polymer physics as well as protein folding dynamics. The viscoelastic response of single polymer chain is difficult and prone to artefacts owing to the complications of hydrodynamics of macroscopic probe itself in the liquid environment. Using a special atomic force microscope, which allows quantitative estimate of viscoelasticity in liquid environments, we measured viscoelastic response of single molecule of Titin. We report here that the dissipation coefficient is below the detection limit of our experiments - with upper bound which is less than reported values in the literature.

biophysics↗

bioRxiv2020

Differential tissue stiffness of body column facilitates locomotion of Hydra on solid substrates

The bell-shaped members of Cnidaria typically move around by swimming, whereas the Hydra polyp can perform locomotion on solid substrates in aquatic environment. To address the biomechanics of locomotion on rigid substrates, we studied the somersaulting locomotion in Hydra. We applied atomic force microscopy to measure the local mechanical properties of Hydras body column and identified the existence of differential Youngs modulus between the shoulder region versus rest of the body column at 3:1 ratio. We show that somersault primarily depends on differential tissue stiffness of the body column and is explained by computational models that accurately recapitulate the mechanics involved in this process. We demonstrate that perturbation of the observed stiffness variation in the body column by modulating the extracellular matrix (ECM) polymerization impairs the somersault movement. These results provide mechanistic basis for the evolutionary significance of differential extracellular matrix properties and tissue stiffness.

biophysics↗

bioRxiv2018

Exocyst subcomplex functions in autophagosome biogenesis by regulating Atg9 trafficking

During autophagy, double membrane vesicles called autophagosomes capture and degrade the intracellular cargo. The de novo formation of autophagosomes requires several vesicle transport and membrane fusion events which are not completely understood. We studied the involvement of Exocyst- an octameric tethering complex, which has a primary function in tethering post-Golgi secretory vesicles to plasma membrane, in autophagy. Our findings indicate not all subunits of exocyst are involved in selective and general autophagy. We show that in the absence of autophagy specific subunits, autophagy arrest is accompanied by accumulation of incomplete autophagosome-like structures. In these mutants, impaired Atg9 trafficking leads to decreased delivery of membrane to the site of autophagosome biogenesis thereby impeding the elongation and completion of the autophagosomes. The subunits of exocyst which are dispensable for autophagic function do not associate with the autophagy specific subcomplex of exocyst.

cell biology↗

bioRxiv2018

Direct Measurement of Dissipation in a Single Protein using Small Amplitude Atomic Force Microscopy

In Krammers theory, stiffness and dissipation coefficient of a protein determine the rate of their conformational change. Using atomic force microscope, it is possible to measure viscoelasticity of a single protein, wherein its dissipative and elastic nature is directly and independently measured. Such measurements are performed, either by measuring the thermal fluctuations of the protein held under a constant force, or by providing small modulations to the protein by dithering the cantilever and measuring its response. In small amplitude approximation, where dither amplitude is comparable to persistence length of polymers, it is possible to measure the proteins viscoelastic response accurately. We measured dissipation in I27 at extremely low pulling speeds ([~] 50 nm/s) and low dither frequencies ([~]100 Hz). At these experimental parameters the dissipation is found to be [~]10-5 kg/s, well above the detection limit of conventional AFM and upper limit predicted by Benedetti et al. Our stiffness data clearly reveals unfolding intermediate of titins individual immunoglobulin units. The intermediate is elongation of folded domains by [~]8 [A], wherein two hydrogen bonds are broken between beta sheets. It was possible to measure this elongation in our experiments. The directly measured internal friction of unfolded polymer chain shows a scaling with tension on the chain. The measurements show that it is possible to measure internal friction in single molecules unambiguously using small amplitude AFM. It suggests that systematic experiments to unravel the relation between directly measured internal friction and folding rates of proteins are possible.

biophysics↗