Biology subjects

Chakrabarti, R.

Publications and source records attributed to Chakrabarti, R..

At least 19 recordsLinked to original sources

bioRxiv2026

FMNL1 polymerized actin filaments complete mitochondrial division downstream of Golgi-derived vesicles

Mitochondrial division includes contributions from several organelles yet the molecular machinery that facilitates the final Drp1-induced scission of the OMM is largely elusive. We elucidate that FMNL1, a formin family actin assembly factor, is recruited to the mitochondrial division sites through the TGN vesicles in a calcium dependent pathway to complete mitochondrial division. FMNL1 is activated specifically by Cdc42 that generates actin filaments to complete division of the OMM in Drp1 and actin-dependent pathway. Depletion of FMNL1 enhances early events in mitochondrial division pathway characterized by enhanced ER-mitochondrial contacts and increased Drp1 and TGN vesicles on mitochondria which still results in failed mitochondrial division.

cell biology↗

bioRxiv2026

A ncBAF to cBAF Switch Regulates Human Hematopoietic Progenitor Myeloid Differentiation

The mammalian BRG-/BRM-associated factor (BAF) chromatin remodeling complexes are crucial regulators of hematopoietic stem and progenitor cell (HSPC) fate. Expression of the ncBAF subcomponent, BRD9, is altered in both normal aging and myeloid malignancies. We find that in adult human HSPC, BRD9 regulates the differentiation of myeloid progenitors. Upon degradation of BRD9 in these cells, ncBAF is displaced from the chromatin, leading in turn to enhanced recruitment of cBAF to bona fide CEBPA target sites. With the shift from ncBAF to cBAF, HSPC differentiation results in increased generation of myeloid-derived suppressor cells (MDSC) that secrete elevated levels of IL-8. Soluble factors from these myeloid cells, which have lost BRD9, enhance the stemness of breast cancer cells in vitro. In contrast, bromodomain inhibition of BRD9 results in minimal alterations of ncBAF binding and myeloid differentiation, indicating that BRD9 acts as a barrier to human MDSC differentiation in a bromodomain-independent manner.

cancer biology↗

bioRxiv2026

An INF2-dependent actin-mediated step in Inositol 1,4,5-trisphosphate receptor cluster formation and activity

Intracellular calcium signaling plays a vital role in regulating various cellular processes including gene regulation, motility, metabolism and cell death. Inositol 1,4,5-trisphosphate receptors (IP3R) on the Endoplasmic Reticulum (ER) are a major cation channel that regulates stimulus-induced calcium release from the ER. While several molecular players regulate activity of IP3R, its regulation by actin filaments were uncharacterized. Here we show that actin filaments polymerized by a specific actin nucleator INF2 facilitates agonist-induced IP3R activity. Our results demonstrate that INF2-mediated actin filaments regulate formation and/or stability of IP3R clusters on the ER that have been previously shown to be hotspots of ER calcium release. Using cell-biological and biochemical techniques we further show that INF2 physically interacts with IP3R isoforms, often at IP3R clusters. While INF2-IP3R interaction is independent of INF2-activity, the ability of INF2 to mediate IP3R clusters is dependent on its actin polymerization activity. Finally, we demonstrate that in addition to its calcium mobilization activity, INF2 on ER specifically regulates IP3R cluster positioning to mediate ER-mitochondrial contacts and facilitate ER to mitochondrial calcium transfer. Overall, these results reveal an actin-dependent step in regulation of IP3R activity both in terms of ER calcium release and modulation of ER-mitochondrial contacts. HighlightsO_LIINF2-mediated actin filaments potentiate agonist-induced IP3R-mediated ER calcium release without affecting the ER calcium stores per se. C_LIO_LIER-localization of INF2 is dispensable for its role on IP3R activity. Moreover INF2-mediated actin filaments affect the activity of all IP3R isoforms. C_LIO_LIINF2 interacts with IP3R in an activity and actin filament independent manner through its C-terminal region. C_LIO_LIINF2 regulates IP3R cluster formation in actin-filament dependent manner and thereby regulates IP3R activity. C_LIO_LIFurther we show that ER-localized INF2 specifically regulate IP3R cluster positioning thereby promoting ER to mitochondrial contact and calcium transfer. C_LI

cell biology↗

bioRxiv2025

Human resistin is critical to activation of the NLRP3 inflammasome in macrophages

Elevated levels of human resistin (hResistin) have been associated with diverse inflammatory diseases, but the precise mechanisms through which hResistins many inflammatory effects contribute to the progression of these diseases remain poorly understood. NLRP3 inflammasome activation is essential in many of these inflammatory conditions; however, there is an unmet explanation connecting hResistin with the NLRP3 inflammatory pathway. Here we describe a unique role of hResistin and its rodent homolog, resistin-like molecule alpha (RELM) in priming and activating the NLRP3 inflammasome. Through qPCR and western blot analysis, we found that hResistin-dependent expression and secretion of high mobility group box 1 (HMGB1) in human macrophages primes the expression of NLRP3, pro-capsase-1, pro-interleukin(IL)-1{beta}, and pro-IL-18. Co-immunoprecipitation showed that hResistin binds to Brutons tyrosine kinase (BTK), which causes the kinase to autophosphorylate, allowing BTK to phosphorylate NLRP3, leading to its assembly and activation with subsequent cleavage of pro-caspase-1, pro-IL-1{beta}, and pro-IL-18, causing initiation of the inflammasome cascade. The hResistin-dependent activation and secretion of IL-1{beta} and IL-18 was critical to the proliferation of human pulmonary vascular smooth muscle cells. For confirmation in vivo, we studied rodent and human pulmonary hypertension (PH). Chronic hypoxia-induced PH in wild-type and RELM KO mice showed RELM-dependent upregulation of HMGB1, BTK, and NLRP3 in mouse lung and was linked to vascular remodeling pathways. Immunohistochemistry revealed that the majority of NLRP3-expressing cells were macrophages and the colocalization of hResistin, BTK, and NLRP3 in macrophages was increased in PH patients lungs. Our work reveals a novel immune mechanism demonstrating hResistin is essential to the priming and activation of NLRP3. Inhibiting NLRP3 activation by blocking hResistin with a human monoclonal antibody suggests a likely therapeutic pathway for NLRP3-driven inflammatory diseases.

immunology↗

bioRxiv2025

Apolipoprotein E reduces the number and activation of non-invariant Natural Killer T cells

Natural Killer T (NKT) cells, which modulate atherosclerosis, include two groups - invariant (iNKT) and variant (vNKT). These subsets differentially regulate the disease progression. Yet, the role of vNKTs in atherosclerosis remains unclear. We induced atherosclerosis by feeding high-fat diet (HFD) to Apoe-/- and analyzed the vNKTs in the liver and spleen. The vNKTs were termed non-iNKTs since they were negatively selected within the NKT population. Available literature suggests NKTs as lipid-recognizing cells; however, to our surprise, the non-iNKT numbers and phenotype remained unchanged between HFD-fed and chow-fed Apoe-/-. This was a blindsiding and unexpected outcome of the non-iNKTs being unaltered and unaffected with or without HFD, indicating no observable impact of atherosclerosis on these subsets. Albeit remaining unperturbed by atherosclerosis, these non-iNKTs demonstrated an identical but unique increase and upregulated activation in both the chow and HFD-fed Apoe-/-. These results instigated an investigation of the baseline correlation of the non-iNKTs between young C57BL/6 (WT) and Apoe-/-. Previously unknown and confounding results revealed upregulated activation and increased non-iNKT numbers but decreased IL-4+ non-iNKTs in the Apoe-/- compared to WT. Furthermore, HFD-fed WT that developed dyslipidemia, elucidated increased hepatic non-iNKTs and splenic IFN-{gamma}+ non-iNKTs compared to chow-fed WT controls. These results were not perceived in chow and HFD-fed Apoe-/-. Although lipid-responsive, non-iNKTs in Apoe-/- mice failed to respond to lipid stress, unlike those in dyslipidemic WT mice. These findings reveal that loss of Apoe, rather than atherosclerosis itself, drives altered non-iNKT biology. Thus, Apoe deficiency intrinsically dysregulates non-iNKTs, masking disease-associated immune changes. Apoe loss alters non-iNKT number and function, independent of atherosclerosis, and challenges the interpretation of immune responses by NKT subsets in Apoe-/- models. Therefore, this study warrants the use of Apoe null mice in studying NKT cells.

immunology↗

bioRxiv2024

Exploring the effect of Hydrocarbon Cross-linkers on the Structure and Binding of Stapled p53 Peptides

Short-length peptides are used as therapeutics due to their high target specificity and low toxicity, for example, peptides designed for targeting the interaction between oncogenic protein p53 and E3 ubiquitin ligase MDM2. These peptide therapeutics form a class of successful inhibitors. To design such peptide-based inhibitors, stapling is one of the methods in which amino acid side chains are stitched together to get conformationally rigid peptides, ensuring effective binding to their partners. In the current work, we use computer simulations to investigate p53 peptides stapled with hydrocarbon chains of different lengths and positions of attachment to the peptide. We subsequently analyze their binding efficiency with MDM2. The introduction of stapling agents restricts the conformational dynamics of peptides, resulting in higher persistence of helicity. The efficiency of the stapling agents has also been verified imposing these stapled peptides to adverse conditions viz. thermal and chemical denaturation. In addition, the conformational exploration of peptides has been investigated using Temperature replica exchange molecular dynamics (T-REMD) simulations. From both the unbiased and T-REMD simulations, p53 with a long hydrocarbon cross-linker shows a more conformationally rigid structure having high helicity compared to other stapled peptides. The rigidity gained due to cross-linking reduces the entropy of the peptide in the free state and thereby facilitates the complexation process. From the binding studies, we have shown that the peptide having multiple short staples has shown a larger enthalpy change during binding free energy, resulting from its orientation and interactions of residues in the binding interface. On the other hand, a peptide with a single long stapling agent shows less entropic penalty than other systems. Our studies suggest a plausible rationale for the relation between the length and the position of attachment of cross-linkers to peptides and the binding free energies between the peptides and their target partners.

biophysics↗

bioRxiv2024

Peri-mitochondrial actin filaments inhibit Parkin assembly via disruption of ER-mitochondrial contact

Mitochondrial damage represents a dramatic change in cellular homeostasis, necessitating metabolic adaptation as well as clearance of the damaged organelle. One rapid response to mitochondrial damage is peri-mitochondrial actin polymerization within 2 mins, which we term ADA (Acute Damaged-induced Actin). ADA is vital for a metabolic shift from oxidative phosphorylation to glycolysis upon mitochondrial dysfunction. In the current study we investigated the effect of ADA on Pink1/Parkin mediated mitochondrial quality control. We show that inhibition of proteins involved in the ADA pathway significantly accelerates Parkin recruitment onto depolarized mitochondria. Addressing the mechanism by which ADA resists Parkin recruitment onto depolarized mitochondria, we found that ADA disrupts ER- mitochondrial contacts in an Arp2/3 complex-dependent manner. Interestingly, over-expression of ER-mitochondrial tethers overrides the effect of ADA, allowing rapid recruitment of not only Parkin but also LC3 after mitochondrial depolarization. During chronic mitochondrial dysfunction, Parkin and LC3 recruitment are completely blocked, which is reversed rapidly by inhibiting ADA. Taken together we show that ADA acts as a protective mechanism, delaying mitophagy following acute damage, and blocking mitophagy during chronic mitochondrial damage.

cell biology↗

bioRxiv2023

Computationally Driven Discovery and Characterization of SIRT3 Activating Compounds that Fully Recover Catalytic Activity under NAD+ Depletion

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to specific substrates, methodologies for the rational design of sirtuin activating compounds -- including compounds that activate mitochondrial sirtuins implicated in the age-related decline of cellular metabolism -- have been lacking. Here, we use computational high-throughput screening methodologies and a biophysical model for activation of the major mitochondrial sirtuin SIRT3 to identify novel small molecule activators of the human SIRT3 enzyme from a 1.2 million compound library. Unlike previously reported SIRT3 activators like Honokiol, which only transiently upregulate SIRT3 under non-steady state conditions and reduce the steady state catalytic efficiency of the enzyme, several of the novel compounds identified here are potent SIRT3 activators in both the steady and non-steady states. Two such compounds can almost double the catalytic efficiency of the enzyme with respect to NAD+, which would be sufficient to almost entirely compensate for the loss in SIRT3 activity that occurs due to the reduction in mitochondrial coenzyme concentration associated with aging, and display AC50s (concentrations of half-maximal activation) as low as 100 nM. The current work thus reports first-in-class, non-allosteric steady state activators that activate SIRT3 through a novel, mechanism-based mode of activation and that may be developed further for therapeutic applications.

biochemistry↗

bioRxiv2022

Mitochondrial dysfunction triggers actin polymerization necessary for rapid glycolytic activation

Mitochondrial damage represents a dramatic change in cellular homeostasis, necessitating rapid responses. One rapid response is peri-mitochondrial actin polymerization, termed ADA (acute damage-induced actin). The consequences of ADA are not fully understood. Here we show that ADA is necessary for rapid glycolytic activation upon inhibition of mitochondrial ATP production in multiple cells, including mouse embryonic fibroblasts and effector CD8+ T lymphocytes, for which glycolysis is an important source of ATP and biosynthetic molecules. Treatments that induce ADA include CCCP, antimycin A, rotenone, oligomycin, and hypoxia. The Arp2/3 complex inhibitor CK666 or the mitochondrial sodium-calcium exchanger (NCLX) inhibitor CGP37157, applied simultaneously with the ADA stimulus, inhibit both ADA and the glycolytic increase within 5-min, suggesting that ADA is necessary for glycolytic stimulation. Two situations causing chronic reductions in mitochondrial ATP production, ethidium bromide treatment (to deplete mitochondrial DNA) and mutation to the NDUFS4 subunit of complex 1 of the electron transport chain, cause persistent peri-mitochondrial actin filaments of similar morphology to ADA. Both peri-mitochondrial actin loss and a 20% ATP decrease occur within 10 min of CK666 treatment in NDUFS4 knock-out cells. We propose that ADA is necessary for rapid glycolytic activation upon mitochondrial impairment, to re-establish ATP production.

cell biology↗

bioRxiv2022

Optogenetics and electron tomography for structure-function analysis of cochlear ribbon synapses

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and transmitter release. Ultrastructurally, the number of docked SVs increased and distances to the presynaptic density decreased upon short (17-25 ms) and long (48-76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results suggest a rapid replenishment of docked SVs at IHC ribbon synapses and argue against synchronized multiquantal release under our experimental conditions.

neuroscience↗

bioRxiv2022

Discovery of novel compounds as potent activators of Sirt3

Among the sirtuin enzymes, Sirt3 is one of the most important deacetylases as it regulates acetylation levels in mitochondria, which are linked to the metabolism of multiple organs and therefore involved in many types of age-related human diseases such as cancer, heart diseases and metabolic diseases. Given the dearth of direct activators of Sirt3, the identification of new modulators could be a key step in the development of new therapeutics. Here we report the discovery of Sirt3 modulators, including activators, through the use of DNA encoded library technology (DEL). The most enriched compounds after DEL selection against SIRT3 were evaluated according to their activity and affinity. Our best activator seems at least as potent as Honokiol (HKL) while the docking studies suggest that our modulators interact with Sirt3 at an atypical site. Our results establish the attractiveness of the DEL technology in identifying novel and potent Sirt3 activators and, therefore, in associated therapeutic applications.

biochemistry↗

bioRxiv2021

DNA amplification in mixed aqueous-organic media: chemical analysis of leading polymerase chain reaction compositions

PCR amplification of GC-rich regions often leads to low yield and specificity. Addition of PCR-enhancing compounds is employed in order to overcome these obstacles. PCR-enhancing additives are low molecular polar organic compounds that are included as undisclosed co-solvents in commercial PCR buffers. In the interest of transparency and to permit further optimization by researchers of PCR compositions for challenging amplification problems, we studied eight PCR buffers by GC/MS to identify and quantify their co-solvents. Buffer specificity, both rich in water and salified substances, required a suitable sample preparation before injection into the GC/MS system. The aqueous phase of each buffer was replaced by an organic solvent to remove, by precipitation and filtration, salified substances which are detrimental to the GC/MS analysis. This approach has demonstrated the advantage of eliminating both water and salified substances without any loss of co-solvents. The sensitivity of the developed method was demonstrated as the main co-solvents were easily detected, identified and quantified. The methodology for identifying the co-solvents is mainly based on comparison of both library matching of acquired MS spectra with NIST library and experimental mass spectra obtained from authentic chemical standards. For the quantification of each co-solvent, deuterated Internal standards of similar structure to the cosolvents were used to correct the variable recovery caused by sample preparation, matrix effects, and ion source variability. The recovery ratio of the developed method was verified and found to be in the range 90-120 %. We then characterized the effects of specific organic co-solvents identified during PCR amplification -- using DNA melting, polymerase thermostability, polymerase activity and real-time PCR methods -- in order to elucidate their mechanism of action and to permit further optimization of their effects on amplification efficiency and specificity.

biochemistry↗

bioRxiv2020

Effect of Stapling on the Thermodynamics of Protein-Peptide Binding

Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. Addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide, the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

biochemistry↗

bioRxiv2020

LncRNA PAINT is Associated with Aggressive Prostate Cancer and Dysregulation of Slug and Related Genes

Long non-coding RNAs (lncRNAs) play regulatory roles in cellular processes and their aberrant expression may drive cancer progression. Here we report the function of a lncRNA PAINT (Prostate Cancer Associated Intergenic Non-Coding Transcript) in promoting prostate cancer (PCa) progression. Upregulation of PAINT was noted in advanced stage and metastatic PCa. Inhibition of PAINT decreased cell proliferation, S-phase progression, increased expression of apoptotic markers, and improved sensitivity to docetaxel and Aurora kinase inhibitor VX-680. Inhibition of PAINT decreased cell migration and reduced expression of Slug and Vimentin. Ectopic expression of PAINT suppressed E-cadherin, increased S-phase progression and cell migration. PAINT expression in PCa cells induced larger colony formation and higher expression of mesenchymal markers. Transcriptome analysis followed by qRT-PCR validation showed differentially expressed genes involved in epithelial mesenchymal transition (EMT), apoptosis and drug resistance in PAINT-expressing cells. Our study establishes an oncogenic function of PAINT in PCa.

cancer biology↗

bioRxiv2020

Nonallosteric Sirtuin Enzyme Activation: Characterization of Hit Compounds

Mammalian sirtuins (SIRT1-SIRT7) are a family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacylases that play critical roles in lifespan and age-related diseases. The physiological importance of sirtuins has stimulated intense interest in designing sirtuin-activating compounds. However, except for allosteric activators of SIRT1-catalyzed reactions that are limited to particular substrates, a general framework for the design of sirtuin-activating compounds has been lacking. Recently, we introduced a general mode of sirtuin activation that is distinct from the known modes of enzyme activation, establishing biophysical properties of small molecule modulators that can, in principle, result in enzyme activation for various sirtuins and substrates. Here, we characterize small molecules reported in the literature to activate the SIRT3 enzyme using a variety of computational, biochemical and biophysical techniques including protein-ligand docking, molecular dynamics simulation, nonlinear reaction dynamics simulation, kinetic assays and thermodynamic assays with multiple substrates and protocols. In particular, we identify the mechanism of action of the compound honokiol on the human SIRT3 enzyme, modeling its effect on active site conformational degrees of freedom and demonstrating how it nonallosterically activates the human SIRT3 enzyme under physiologically relevant conditions. We show that honokiol constitutes a hit compound for the design of a new generation of nonallosteric activators that can activate SIRT3 through the proposed mechanism-based mode of activation.

biophysics↗

bioRxiv2020

Mechanism-based enzyme activating compounds

Compared to inhibitors, which constitute the vast majority of todays drugs, enzyme activators have considerable advantages, especially in the context of enzymes that regulate reactive flux through metabolic pathways associated with chronic, age-related diseases and lifespan. Across all families of enzymes, only a dozen or so distinct classes of small molecule activators have been characterized. Enzyme activators that are not based on naturally evolved allosteric mechanisms are much more difficult to design than inhibitors, because enzymatic catalysis has been optimized over billions of years of evolution. Here, we introduce modes of enzyme activation based on the catalytic reaction mechanisms of enzymes for which naturally evolved activators may not exist. We establish biophysical properties of small molecule modulators that are necessary to achieve desired changes in the steady state and non-steady state parameters of these enzymes, including changes in local conformational degrees of freedom conducive to the enhancement of catalytic activity that can be identified through computational modeling of their active sites. We illustrate how the modes of action of several compounds reported to activate enzymes without known allosteric sites may be understood using the framework presented. We also present simulations and new experimental results in support of this framework, including identification of the mechanism of a compound that activates the human SIRT3 enzyme, which does not contain a known allosteric site, under physiologically relevant conditions.

biochemistry↗

bioRxiv2019

Two distinct actin filament populations have effects on mitochondria, with differences in stimuli and assembly factors

Recent studies show that mitochondria and actin filaments work together in two contexts: 1) increased cytoplasmic calcium induces cytoplasmic actin polymerization that stimulates mitochondrial fission, and 2) mitochondrial depolarization causes actin assembly around mitochondria, with roles in mitophagy. It is unclear whether these two processes utilize similar actin assembly mechanisms. Here, we show that these are distinct actin assembly mechanisms in the acute phase after treatment (<10 min). Calcium-induced actin assembly is INF2-dependent and Arp2/3 complex-independent, whereas depolarization-induced actin assembly is Arp2/3 complex-dependent and INF2-independent. The two types of actin polymerization are morphologically distinct, with calcium-induced filaments throughout the cytosol and depolarization-induced filaments as \"clouds\" around depolarized mitochondria. We have previously shown that calcium-induced actin stimulates increases in both mitochondrial calcium and recruitment of the dynamin GTPase Drp1. In contrast, depolarization-induced actin is temporally-associated with extensive mitochondrial dynamics that do not result in mitochondrial fission, but in circularization of the inner mitochondrial membrane (IMM). These dynamics are dependent upon the protease Oma1 and independent of Drp1. Actin cloud inhibition causes increased IMM circularization, suggesting that actin clouds limit these dynamics.\n\nSummary statementMitochondrial depolarization induces Arp2/3 complex-dependent actin clouds that restrain mitochondrial shape changes induced by Oma1 on the inner mitochondrial membrane. A distinct actin network stimulates mitochondrial fission in response to calcium.

cell biology↗

bioRxiv2019

Internal friction can be measured with the Jarzynski equality

A simple protocol for the extraction of the internal friction coefficient of polymers is presented. The proposed scheme necessitates repeatedly stretching the polymer molecule, and measuring the average work dissipated in the process by applying the Jarzynski equality. The internal friction coefficient is then estimated from the average dissipated work in the hypothetical limit of zero solvent viscosity. The validity of the protocol is established through Brownian dynamics simulations of a single-mode spring-dashpot model for a polymer. Well-established single-molecule manipulation techniques, such as optical tweezer-based pulling, can be used to implement the suggested protocol experimentally.

biophysics↗