Biology subjects

Pullarkat, P. A.

Publications and source records attributed to Pullarkat, P. A..

8 recordsLinked to original sources

bioRxiv2025

Calcium chelation promotes microtubule regrowth and axonal recovery after laser-induced axonal injury

Traumatic stretch or crush injury to axons causes widespread and often irreversible damage to the axonal cytoskeleton, in which calcium-mediated breakdown is known to play a central role. Unlike complete transection, where recovery must proceed through formation of a new growth cone, milder injury can disrupt the axonal cytoskeleton while leaving the plasma membrane intact. How the cytoskeleton fails, and how it can recover, under these conditions remains unclear. Here we address this using a partial laser-ablation method that damages the cytoskeleton and evokes a calcium transient while preserving membrane continuity. We show that the ensuing cytoskeletal retraction is set by a mechanical balance between acto-myosin contractility and microtubule stability: stabilizing microtubules or inhibiting acto-myosin contractility suppresses retraction. Moreover, chelating extracellular calcium mitigates degeneration and, in a subset of axons, permits complete recovery. We also show that microtubules and actin filaments show distinct loss and recovery dynamics and provide a hypothesis for the "burning-fuse" -like depolymerization of the microtubule bundle. These findings provide insights into how the axonal cytoskeleton collapses and recovers after injury and suggest strategies for mitigating damage.

neuroscience↗

bioRxiv2025

Estimating anisotropy of single cells using a compliant biaxial stretcher

Understanding the mechanical properties of single cells is essential for elucidating their responses to mechanical cues and plays a critical role in development, disease progression, and mechanotransduction. However, probing these properties at the single-cell level remains technically challenging. Traditional methods for probing cellular mechanics are often restricted to a single deformation mode and rely on complex instrumentation, limiting their adaptability. Hence, no single platform currently offers multi-modal cell manipulation, which motivates the development of more versatile tools. Towards this, we are developing an ensemble of compliant micromechanisms that enables diverse modes of cell manipulation on a single chip. Building on our earlier work with a uniaxial stretcher, here we demonstrate estimation of cellular anisotropy using a biaxial stretcher. Since the mechanism transforms motion using deformation, cell stretching forces can be estimated through image-based displacement measurements, without additional sensing modalities, considerably simplifying the device fabrication and operation. By assembling these uniaxial and biaxial stretching micromechanisms on a single chip, we can establish a multi-modal manipulation platform for single cells, which can be augmented with shearing and twisting modes in the future. Such a simple and versatile platform presents a paradigm shift in mechanical testing of single cells.

bioengineering↗

bioRxiv2025

Investigation of Axonal Beading Induced by Photo-oxidation

Oxidative stress caused by the presence of excessive free radicals (reactive oxygen species) is known for its adverse effects in cellular function. When initiated by fluorescent dye excitation, it can lead to photo-oxidative stress, causing cellular damage. In this study, we investigate axonal beading induced by the photo-oxidation of a membrane based fluorescent dye. Our findings show that apart from the use of oxygen scavengers, chelation of free calcium and stabilizing microtubules or actin filaments by pharmacological agents can mitigate oxidative stress induced axonal beading. Furthermore, we take advantage of this light induced oxidative stress phenomenon to probe axonal response to spatially confined perturbations of varying strength. We demonstrate that low excitation levels lead to long-lived localized beading, whereas higher levels of excitation result in global degeneration and beading of axons. Besides the results presented here, this light based technique is a convenient tool to investigate the effect of local oxidative stress at the tissue level, for example, in cultures of brain slices or organoids.

neuroscience↗

bioRxiv2022

Cell adhesion strength and tractions are mechano-diagnostic features of cellular invasiveness

The adhesion of cells to substrates occurs via integrin clustering and binding to the actin cytoskeleton. Oncogenes modify anchorage-dependent mechanisms in cells during cancer progression. Fluid shear devices provide a label-free, non-invasive way to characterize cell-substrate interactions and heterogeneities in the cell populations. We quantified the critical adhesion strengths of MCF7, MDAMB-231, A549, HPL1D, HeLa, and NIH3T3 cells using a custom fluid shear device. The detachment response was sigmoidal for each cell type. A549 and MDAMB-231 cells had significantly lower adhesion strengths at {tau}50 than their non-invasive counterparts, HPL1D and MCF7. Detachment dynamics was inversely correlated with cell invasion potentials. A theoretical model, based on {tau}50 values and the distribution of cell areas on substrates, provided good fits to data from de-adhesion experiments. Quantification of cell tractions, using the Reg-FTTC method on 10 kPa polyacrylamide gels, showed highest values for invasive, MDAMB-231 and A549, cells compared to non-invasive cells. Immunofluorescence studies show differences in vinculin distributions: non-invasive cells have distinct vinculin puncta, whereas invasive cells have more dispersed distributions. The cytoskeleton in non-invasive cells was devoid of well-developed stress fibers, and had thicker cortical actin bundles in the boundary. These correlations in adhesion strengths with cell invasiveness, demonstrated here, may be useful in cancer diagnostics and other pathologies featuring misregulation in adhesion.

bioengineering↗

bioRxiv2019

The roles of microtubules and membrane tension in axonal beading, retraction, and atrophy

Axonal beading--formation of a series of swellings along the axon--and retraction are commonly observed shape transformations that precede axonal atrophy in Alzheimers, Parkinson, and other neurodegenerative conditions. The mechanisms driving these morphological transformations are poorly understood. Here we report controlled experiments which can induce either beading or retraction and follow the time evolution of these responses. By making quantitative analysis of the shape modes under different conditions, measurement of membrane tension, and using theoretical considerations, we argue that membrane tension is the main driving force that pushes cytosol out of the axon when microtubules are degraded, causing axonal thinning. Under pharmacological perturbation, atrophy is always retrograde and this is set by a gradient in the microtubule stability. The nature of microtubule depolymerization dictates the type of shape transformation vis a vis beading or retraction. Elucidating the mechanisms of these shape transformations will facilitate development of strategies to prevent or arrest axonal atrophy due to neurodegenerative conditions.

biophysics↗

bioRxiv2019

The axonal actin-spectrin lattice acts as shock absorbers to protect neurons from stretch-induced damage

Axons are thin tubular extensions generated by neuronal cells to transmit signals across long distances. In the peripheral and the central nervous systems, axons experience large deformations during normal activity or as a result of injury. Yet, axon biomechanics, and its relation to the internal structure that allows axons to withstand such deformations, is poorly understood. Up to now, it has been generally assumed that microtubules and their associated proteins are the major load-bearing elements in axons. We revise this view point by combining mechanical measurements using a custom developed force apparatus with biochemical or genetic modifications to the axonal cytoskeleton, revealing an unexpected role played by the actin-spectrin skeleton. For this, we first demonstrate that axons exhibit a reversible strain-softening response, where its steady state elastic modulus decreases with increasing strain. We then explore the contributions from the various cytoskeletal components of the axon, and show that the recently discovered membrane-associated skeleton consisting of periodically spaced actin filaments interconnected by spectrin tetramers play a prominent mechanical role. Finally, using a theoretical model we argue that the actin-spectrin skeleton act as an axonal tension buffer by reversibly unfolding repeat domains of the spectrin tetramers to buffer excess mechanical stress.

biophysics↗

bioRxiv2017

Cytoskeletal mechanisms of axonal contractility

Mechanotransduction is likely to be an important mechanism of signalling in thin, elongated cells like neurons. Maintenance of pre-stress or rest tension may facilitate mechanotransduction in these cells. In recent years, functional roles for mechanical tension in neuronal development and physiology are beginning to emerge but the cellular mechanisms regulating neurite tension remain poorly understood. Active contraction of neurites is a potential mechanism of tension regulation. In this study, we have explored cytoskeletal mechanisms mediating active contractility of neuronal axons. We have developed a simple assay where we evaluate contraction of curved axons upon trypsin-mediated detachment. We show that curved axons undergo contraction and straighten upon de-adhesion. Axonal straightening was found to be actively driven by actomyosin contractility, while microtubules may subserve a secondary role. We find that while axons show a monotonous decrease in length upon contraction, subcellularly, the cytoskeleton shows a heterogeneous contractile response. Further, using an assay for spontaneous development of tension without trypsin-induced de-adhesion, we show that axons are intrinsically contractile. These experiments, using novel experimental approaches, implicate the axonal cytoskeleton in tension homeostasis. Our data suggest that while globally the axon behaves as a mechanical continuum, locally the cytoskeleton is remodelled heterogeneously.

biophysics↗

bioRxiv2017

Quantifying cell adhesion dynamics using a custom-made fluid shear device: experiments and model

Changes in cell-substrate adhesion are believed to signal the onset of cancer metastasis, but such changes must be quantified against background levels of intrinsic heterogeneity between cells. Variations in cell-substrate adhesion strengths can be probed through biophysical measurements of cell detachment from substrates upon the application of an external force. Here, we investigate, theoretically and experimentally, the detachment of cells adhered to substrates when these cells are subjected to fluid shear. We present a theoretical framework within which we calculate the fraction of detached cells as a function of shear stress for fast ramps as well as for the decay in the fraction of detached cells at fixed shear stress as a function of time. Using HEK and 3T3 fibroblast cells as experimental model systems, we extract characteristic force scales for cell adhesion as well as characteristic detachment times. We estimate force-scales of ~ 500 pN associated to a single focal contact, and characteristic time-scales of 190 [≤]{tau} [≤] 350s representing cell-spread-area dependent mean first passage times to the detached state at intermediate values of the shear stress. Variations in adhesion across cell types are especially prominent when cell detachment is probed by applying a time-varying shear stress. These methods can be applied to characterizing changes in cell adhesion in a variety of contexts, including metastasis.

biophysics↗