Biology subjects

Qamar, S.

Publications and source records attributed to Qamar, S..

12 recordsLinked to original sources

bioRxiv2025

Mapping high resolution, multidimensional phase diagrams of physiological protein condensates

Biomolecular condensates are membraneless compartments, crucial for organising and regulating diverse cellular processes. Current approaches to study condensate biology either use simplified recombinant protein systems with limited physiological relevance, or complex live-cell models with restricted experimental control and scalability. Here, we present ExVivo PhaseScan, a droplet microfluidics platform that couples mammalian lysate-based reconstitution with scalable analysis to generate high-resolution phase diagrams of physiological protein condensates. We apply this approach to study two complex multicomponent condensate systems, stress granules and nucleoli, and dissect the physicochemical interactions that influence their stability. We further developed a machine learning pipeline to analyse condensate morphology which we use to reveal how mutations in the ALS-linked protein FUS remodels condensate phase landscapes. We identify liquid-to-solid transitions of mutant FUS within stress granules and nucleoli, and show that these transitions can be reversed by RNA aptamer-based interventions. Together, these findings establish ExVivo PhaseScan as a versatile tool for dissecting the physicochemical and pathological regulation of condensates, with potential to inform therapeutic strategies for diseases driven by aberrant phase transitions.

biophysics↗

bioRxiv2025

Spike protein E2 of chikungunya virus: a plant-based vaccine exhibited potent immunogenicity in BALB/c mice

Over the last two decades, chikungunya virus (CHIKV) infections have surged worldwide, causing significant suffering. CHIKV E2 gene codes for spike protein E2, essential for virus-host interactions and thus serves as a potential vaccine candidate. We expressed a full-length E2 (S27 African prototype) in both E. coli and Nicotiana tabacum, which triggered an immune response in BALB/c mice. First, E2 was computationally analyzed for PTM patterns and prediction of B-cell and T-cell epitopes. Next, molecular docking of epitopes with MHCs (Class I and II) revealed high affinity, confirmed by Molecular Dynamics Simulation. Then, the chemically synthesized E2-6xHis tag was cloned and expressed in both E. coli and N. tabacum under T7 and CaMV promoters, respectively. E2 was cloned into the pUC57 vector (E. coli) and expressed using the pET28a(+) vector in BL21(DE3)pLysS cells, followed by cloning into the pCAMBIA1302 vector and transformation into Agrobacterium tumefaciens. RT-PCR and confocal visualization confirmed the formation of E2 transcripts. Recombinant E2 was purified on Ni-NTA columns and visualized as a protein of [~]49 kDa on SDS PAGE. Finally, E2 was injected into BALB/c mice; neutralizing antibodies, including IgG, were detected as positive in the indirect ELISA, with the highest levels observed at 3 days post-infiltration (3 dpi). Western blot also confirmed E2 expression in E coli and tobacco, and induction of E2-specific antibodies in BALB/c mice. This study presents a promising approach to developing a safe and effective vaccine against chikungunya fever in plants.

molecular biology↗

bioRxiv2025

DNA Partitioning Modulates Liquid-to-Solid Transitions and the Internal Microstructure of FUS Condensates

Protein liquid-liquid phase separation has recently been recognized as an essential process involved in cellular functions, including transcription, translation, and DNA damage repair. However, a further liquid-to-solid transition (LST) of condensates due to mutation or external stimuli can result in aggregation, sometimes pathological. The unique ability of protein condensates to concentrate and sequester biomolecules is at the heart of the regulatory mechanism, controlling the dynamics and function of the condensates. While the recruitment of essential biomolecules, such as RNA has been studied to have the impact to the condensate formation, how DNA partition can affect the phase behaviour and dynamics of protein condensates is not fully elucidated. In this study, we investigate both the short-term and long-term kinetics of double-stranded DNA partitioning into preformed fresh and aged FUS protein condensates. Confocal imaging shows that DNA partition follows the core-shell diffusion pattern within the condensates. LST slows down and reduces FUS condensates ability to recruit DNA but stabilizes the DNA-FUS condensate complex due to the heterogeneous solid network formation. Using the optical technique of Spatial Dynamic Mapping (SDM), we find that DNA partition promotes coalescence and alters the characteristics of the condensates. The partition made the condensates more dynamic in the short term (within minutes) but accelerated LST in long-term incubation (within hours), ultimately leading to an irreversible porous core-shell structure of FUS condensates. Our findings reveal the kinetics of DNA partition during aging and its impact on LST, underlining the modulation of condensate properties by molecule sequestration, shedding light on possible regulation of disease-related LST of biomolecular condensates.

biophysics↗

bioRxiv2025

Scaling relations of multi-component phase coexistence boundaries

Multicomponent phase-separating molecular systems play a key role as membraneless organelles in living cells. Phase diagrams are indispensable for probing the concentration-dependent behaviour of these condensates, yet their interpretation has remained largely qualitative due to the challenges of modelling complex, multicomponent interactions. Here, we present a generic framework for quantitative analysis of phase diagrams. We derive an analytical expression for the phase boundary normal vector, which reveals two distinct scaling regimes: one for associative phase separation, characterised by a power-law scaling analogous to mass-action kinetics that allows for direct extraction of dense phase solute molar ratios, and another for condensate dissolution, where an exponential scaling quantifies three-body repulsion effects triggered by the addition of a new component. We demonstrate the practical utility of our framework by applying it to a range of experimentally measured phase diagrams, including those for proteins such as NSP5, NSP2, FUS, Whi3, G3BP1, and antimicrobial peptides. Collectively, our work not only provides a clear quantitative interpretation of phase diagrams but also opens new avenues for the rigorous characterisation of multicomponent phase-separating systems.

biophysics↗

bioRxiv2024

Temperature-induced changes in protein interactions control RNA recruitment to G3BP1 condensates

Biomolecular condensates have emerged as prominent regulators of dynamic subcellular organisation and essential biological processes. Temperature, in particular, exerts a significant influence on the formation and behaviour of biomolecular condensation. For example, during cellular heat stress, stress granules (SGs) are formed from RNA-binding proteins (RBPs) and RNA, forming liquid condensates to protect the RNA from damage. However, the molecular mechanisms leading to changes in protein phase behaviour are not well understood. To answer how temperature modulates protein interactions and phase behaviour, we developed a high-throughput microfluidic platform, capable of mapping the phase space and quantifying protein interactions in a temperature-dependent manner. Specifically, our approach measures high-resolution protein phase diagrams as a function of temperature, while accurately quantifying changes in the binodal, condensate stoichiometry and free energy contribution of a solute, hence, providing information about the underlying mechanistic driving forces. We employ this approach to investigate the effect of temperature changes on the phase separation of the stress granule scaffold protein Ras GTPase-activating protein-binding protein 1 (G3BP1) with PolyA-RNA. Surprisingly, we find that the G3BP1/RNA phase boundary remains unaffected by the increasing temperature but the underlying stoichiometry and energetics shift, which can only be revealed with high-resolution phase diagrams. This indicates that temperature-induced dissolution is counteracted by entropic processes driving phase separation. With increasing temperature, the G3BP1 content in condensates decreases alongside with a reduction of the free energy of protein interactions, while the RNA content increases driven by entropically favoured hydrophobic interactions. In the context of cellular heat SG formation, these findings could indicate that during heat shock, elevated temperatures directly induce RNA recruitment to stress granules as a cytoprotective mechanism by finetuning the strength of protein and RNA interactions.

biophysics↗

bioRxiv2023

Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates

In addition to the native state, proteins can form liquid-like condensates, viscoelastic condensates, such as gels, as well as solid-like condensates, such as amyloid fibrils, crystals and amorphous materials. The material properties of these condensates play important roles in their cellular functions, with aberrant liquid-to-solid phase transitions having been implicated in neurodegenerative diseases. However, the molecular changes and resultant material properties across the whole phase space of condensates are complex and yet to be fully understood. The extreme sensitivity to their environment, which enables their biological function, is also what makes protein condensates particularly challenging experimental targets. Here, we provide a characterisation of the ageing behaviour of the full-length fused in sarcoma (FUS) protein. We achieve this goal by using a microfluidic sample deposition technology to enable the application of surface-based techniques to the study of biological condensates. We first demonstrate that we maintain relevant structural features of condensates in physiologically-relevant conditions on surfaces. Then, using a combination of atomic force microscopy and vibrational spectroscopy, we characterise the spatio-temporal changes in the structure and mechanical properties of the condensates to reveal local phase transitions in individual condensates. We observe that initially dynamic, fluid-like condensates undergo a global increase in elastic response conferred by an increase in the density of cation-{pi} intermolecular interactions. Solid-like structures form first at condensate-solvent interfaces, before heterogeneously propagating throughout the aged fluid core. These solid structures are composed of heterogenous, non-amyloid {beta}-sheets, which are stabilised by hydrogen-bonding interactions not observed in the fluid state. Overall, this study identifies the molecular conformations associated with different physical states of FUS condensates, establishing a technology platform to understand the role of phase behaviour in condensate function and dysfunction.

biophysics↗

bioRxiv2023

Linking modulation of bio-molecular phase behaviour with collective interactions

Bio-molecular condensates formed in the cytoplasm of cells are increasingly recognised as key spatiotemporal organisers of living matter, and are implicated in a wide range of functional or pathological processes. This discovery opens up a new avenue for condensate-based applications and a crucial step in controlling this process is to understand the underlying interactions driving condensate formation or dissolution. However, these condensates are highly multi-component assemblies and many inter-component interactions are present, rendering it difficult to identify key promoters of phase separation. In this work, we extend the recently formulated dominance analysis to modulations of condensate formation. By carrying out dilute phase concentration measurements of a single target solute, the theoretical framework allows one to deduce whether the modulator acts on the target solute or another unspecified, auxiliary solute, as well as the attractive/repulsive nature of the added interaction. This serve as a general guide towards deducing possible modulation mechanisms on the molecular level, which can be complemented by orthogonal measurements. As a case study, we investigate the modulation of G3BP1/RNA condensates by the small molecule suramin, and the dominance measurements point towards a dissolution mechanism where suramin acts on G3BP1 to disrupt G3BP1/RNA interactions, as confirmed by a diffusional sizing assay. Our approach and the dominance framework have a high degree of adaptability and can be applied in many other condensate-forming systems.

biophysics↗

bioRxiv2023

A hybrid CNN-Random Forest algorithm for bacterial spore segmentation and classification in TEM images

We present a new approach to segment and classify bacterial spore layers from Transmission Electron Microscopy (TEM) images using a hybrid Convolutional Neural Network (CNN) and Random Forest (RF) classifier algorithm. This approach utilizes deep learning, with the CNN extracting features from images, and the RF classifier using those features for classification. The proposed model achieved 73% accuracy, 64% precision, 46% sensitivity, and 47% F1-score with test data. Compared to other classifiers such as AdaBoost, XGBoost, and SVM, our proposed model demonstrates greater robustness and higher generalization ability for non-linear segmentation. Our model is also able to identify spores with a damaged core as verified using TEMs of chemically exposed spores. Therefore, the proposed method will be valuable for identifying and characterizing spore features in TEM images, reducing labor-intensive work as well as human bias.

microbiology↗

bioRxiv2023

ANXA11 biomolecular condensates facilitate protein-lipid phase coupling on lysosomal membranes

Phase transitions of cellular proteins and lipids play a key role in governing the organisation and coordination of intracellular biology. The frequent juxtaposition of proteinaceous biomolecular condensates to cellular membranes raises the intriguing prospect that phase transitions in proteins and lipids could be co-regulated. Here we investigate this possibility in the ribonucleoprotein (RNP) granule-ANXA11-lysosome ensemble, where ANXA11 tethers RNP granule condensates to lysosomal membranes to enable their co-trafficking. We show that changes to the protein phase state within this system, driven by the low complexity ANXA11 N-terminus, induce a coupled phase state change in the lipids of the underlying membrane. We identify the ANXA11 interacting proteins ALG2 and CALC as potent regulators of ANXA11-based phase coupling and demonstrate their influence on the nanomechanical properties of the ANXA11-lysosome ensemble and its capacity to engage RNP granules. The phenomenon of protein-lipid phase coupling we observe within this system offers an important template to understand the numerous other examples across the cell whereby biomolecular condensates closely juxtapose cell membranes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=177 SRC="FIGDIR/small/533832v1_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@16f3e10org.highwire.dtl.DTLVardef@538f39org.highwire.dtl.DTLVardef@1e00d4eorg.highwire.dtl.DTLVardef@89967d_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

Mutation of the ALS/FTD-associated RNA-binding protein FUS alters axonal cytoskeletal organisation

Aberrant condensation and localisation of the RNA-binding protein fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is also associated with cytoskeletal defects, genetically and through observations of compromised axonal transport. Here, we asked whether compromised axonal cytoskeletal organisation is an early feature of FUS-associated ALS/FTD. We used an ALS-associated mutant FUS(P525L) and the FTD-mimic hypomethylated FUS, FUS(16R), to investigate the common and distinct cytoskeletal changes found in these two reported Xenopus models. Combining a novel atomic force microscopy (AFM)-based approach for in vitro cytoskeletal characterisation and in vivo axonal branching analysis, we found that mutant FUS reduced actin density in the dynamically remodelling growth cone, and reduced axonal branch complexity. We furthermore found evidence of an axon looping defect for FUS(P525L). Therefore, we show that compromised actin remodelling is potentially an important early event in FUS-associated pathogenesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/510780v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1e9a92aorg.highwire.dtl.DTLVardef@161076eorg.highwire.dtl.DTLVardef@12edf0forg.highwire.dtl.DTLVardef@365785_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Multiphase condensates from a kinetically arrested phase transition

The formation of biomolecular condensates through liquid-liquid phase separation from proteins and nucleic acids is emerging as a spatial organisational principle used by living cells. Many such biomolecular condensates are not, however, homogeneous fluids, but contain an internal structure consisting of distinct sub-compartments with different compositions. In many instances, such compartments inside the condensate are depleted in the biopolymers that make up the condensate. Here, we describe that this multiphase structure arises from a kinetically arrested phase transition. The combination of a change in composition coupled with a slow response to this change can lead to the spontaneous formation of multiple emulsions consisting of several inner cores within a polymer-rich middle phase. In the case of liquid-like biomolecular condensates, the slow diffusion of biopolymers causes nucleation of biopolymer-poor liquid inside of the condensate to achieve the new equilibrium composition. This framework shows that multiphase condensates can be a result of kinetic trapping, rather than thermodynamic stability, and provides and avenue to understand and control the internal structure of condensates in vitro and in vivo.

biophysics↗

bioRxiv2020

Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquid-liquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.

biophysics↗