Biology subjects

Subramaniam, A. B.

Publications and source records attributed to Subramaniam, A. B..

6 recordsLinked to original sources

bioRxiv2025

Polymer-assisted assembly of giant vesicles in physiological solutions

Thin film hydration methods are widely used to assemble giant unilamellar vesicles (GUVs), but their efficiency drops sharply in solutions of physiological ionic strength due to enhanced adhesion between stacked lipid bilayers, which suppresses bud and foam-like mesophase formation. Here, we introduce polymer-coated nanocellulose paper (PCP) as a nanostructured substrate that overcomes this limitation. Hydration of lipid films on PCP--termed PCP-assisted hydration-- achieves high GUV yields across a broad temperature range (4-45 {degrees}C) using diverse soluble polymers, including ultralow-gelling agarose, hyaluronic acid, dextran, carrageenan, and polylysine. The nanoscale curvature of the cellulose fibers and the osmotic pressure generated by polymer dissolution act synergistically to promote membrane budding even under physiological salt conditions. The approach is scalable--supporting GUV production from millimeter-sized pieces to large-format sheets--and biocompatible, enabling encapsulation of complex biochemical systems such as cell-free expression mixtures and actin-fascin assemblies. PCP-assisted hydration thus provides a robust, versatile, and high-yielding platform for generating functional GUVs under physiological conditions.

biophysics↗

bioRxiv2024

Insights into a clock's fidelity through vesicular encapsulation

The single-celled cyanobacterium, Synechococcus elongatus, generates circadian rhythms with exceptional fidelity and synchrony despite their femtoliter volumes. Here, we explore the mechanistic aspects of this fidelity, by reconstituting the KaiABC post-translational oscillator (PTO) in cell-mimetic giant vesicles (GUVs) under well-defined conditions in vitro. PTO proteins were encapsulated with a coefficient of variation that closely matched protein variations observed in live cells. Using fluorescently labeled KaiB and confocal microscopy, we were able to measure circadian rhythms generated by thousands of encapsulated PTOs at the single-vesicle level for several days as a function of protein concentration and GUV size. We find that PTO fidelity decreased with decreasing levels of encapsulated PTO proteins and in smaller GUVs. We also observed that in encapsulated PTOs, a significant fraction of KaiB localized to GUV membranes like it does in cyanobacteria. A mathematical model that uses empirical bulk concentration and stoichiometry limitations suggests that cyanobacteria overcome challenges to fidelity by expressing high levels of PTO proteins along with the CikA and SasA proteins, which buffer stochastic variations in the levels of KaiA and KaiB, respectively. Further, the model suggests that the transcription-translation feedback loop (TTFL) contributes at most a small percentage to the overall fidelity of the cyanobacterial circadian clock under constant conditions but is essential for maintaining phase synchrony. Our results are the first experimental demonstration of populations of synthetic cells that can autonomously keep circadian time. Additionally, the approach of using bulk relationships to understand complex phenomena in cell-like systems could be useful for understanding other collective behavior important in biology, such as liquid-liquid phase separation.

biophysics↗

bioRxiv2024

Ultrahigh yields of giant vesicles obtained through mesophase evolution and breakup

Self-assembly odry amphiphilic lipid films on surfaces upon hydration is a crucial step in the formation of cell-like giant unilamellar vesicles (GUVs). GUVs are useful as model biophysical systems, as chassis for synthetic biology, and in biomedical applications. Here via combined quantitative measurements of the molar yield and distributions of sizes and high-resolution imaging of the evolution of lipid films on surfaces, we report the discovery of a previously unknown pathway for the assembly of GUVs which can lead to ultrahigh yields of > 50 %. This yield is about 60 % higher than any GUV yield reported to date. The "shear-induced fragmentation" pathway occurs in membranes containing 3 mol % of the poly(ethylene glycol) modified lipid PEG2000-DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]), when a lipid-dense foam-like mesophase forms upon hydration. The membranes in the mesophase fragment and close to form GUVs upon application of fluid shear. Experiments with varying mol % of PEG2000-DSPE and with lipids with partial molecular similarity to PEG2000-DSPE show that ultrahigh yields are only achievable under conditions where the lipid-dense mesophase forms. The increased yield of GUVs compared to mixtures without PEG2000-DSPE was general to other flat supporting surfaces such as stainless steel sheets and to various lipid mixtures. Since FDA-approved liposomal and lipid nanoparticle formulations use PEG2000-DSPE, these results provide a useful route to obtaining ultrahigh yields of GUVs that are suitable for biomedical applications.

biophysics↗

bioRxiv2023

Dynamics of giant vesicle assembly from thin lipid films

Giant unilamellar vesicles (GUVs) are micrometer-scale lipid assemblies that emulate key characteristics of biological cell membranes. GUVs can be obtained when solid-supported thin films of lipids are hydrated in aqueous solutions. However, a comprehensive understanding of their assembly dynamics has been lacking, impeding mechanistic insights. Here, we report the time dependence of the distribution of sizes and molar yield of GUVs obtained through a novel stopped-time technique. We compare three commonly used techniques, PAPYRUS (Paper-Abetted amPhiphile hYdRation in aqUeous Solutions) gentle hydration, and electroformation. We demonstrate that all three techniques show sigmoidal yield curves. Yields increase monotonically before reaching a plateau, with surprisingly high yields 60 seconds after hydration. Gentle hydration shows limited time evolution in contrast to PAPYRUS and electroformation. Exploration of bud dynamics on the surfaces uncovers bud emergence, diameter growth, and merging phenomena. To provide a comprehensive explanation of our observations, we employ the thermodynamic budding and merging model. This work expands our understanding of GUV assembly dynamics and offers fundamental insights into the underlying thermodynamic principles governing this process.

biophysics↗

bioRxiv2022

Osmotic pressure enables high yield assembly of giant vesicles in solutions of physiological ionic strengths

Giant unilamellar vesicles (GUVs) are micrometer scale minimal cellular mimics that are useful for synthetic biology and drug delivery. Unlike assembly in low-salt solutions, assembly of GUVs in solutions with ionic concentrations of 100-150 mM Na/KCl (salty solutions) is challenging. Chemical compounds deposited on the substrate or in the lipid mixture could assist in the assembly of GUVs. Here, we investigate quantitatively the effects of temperature and chemical identity of six assisting polymeric and small molecule compounds on the molar yields of GUVs composed of three different lipid mixtures using high resolution confocal microscopy and large dataset image analysis. All the polymers moderately increased the yields of GUVs either at 22 or 37 degrees Celsius, whereas the small molecule compound was ineffective. Low gelling temperature agarose is the singular compound that consistently produces yields of GUVs of greater than 10 %. We propose a free energy model of budding to explain the effects of polymers in assisting the assembly of GUVs. The osmotic pressure exerted on the membranes by the dissolved polymer balances the increased adhesion between the membranes, thus reducing the free energy for bud formation. Data obtained by modulating the ionic strength and ion valency of the solution shows that the evolution of the yield of GUVs support our models prediction. In addition, polymer specific interactions with the substrate and the lipid mixture effects yields. The uncovered mechanistic insight provides a quantitative experimental and theoretical framework to guide future studies. Additionally, this work shows a facile means for obtaining GUVs in solutions of physiological ionic strengths.

biophysics↗

bioRxiv2020

Nanoscale curvature promotes high yield spontaneous formation of cell-mimetic giant vesicles

To date, surface-assisted assembly of cell-like giant vesicles use planar surfaces and require the application of electric fields or dissolved molecules to obtain adequate yields. Here, we present the use of nanoscale surface curvature and hydrophilic surface chemistry to promote the high yield assembly of GUVs. We show that assembly on surfaces composed of entangled hydrophilic nanocellulose fibers results in an unprecedented 100,000-fold reduction in costs while increasing yields compared to extant techniques. Quantitative measurements of yields provide mechanistic insight on the effect of nanoscale curvature and the effect of surface chemistry. We present a thermodynamic budding and merging, BNM, model that unifies observations of assembly. The BNM model considers the change in free energy by balancing elastic, adhesion, and membrane edge energies in the formation of surface-attached spherical buds. Due to curvature and the hydrophilicity of cellulose, energetically unfavorable formation of buds on planar and spherical surfaces becomes favorable (spontaneous) on surfaces composed of cylindrical cellulose nanofibers. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/227686v2_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@3452a4org.highwire.dtl.DTLVardef@11da2a1org.highwire.dtl.DTLVardef@a021a1org.highwire.dtl.DTLVardef@1000acb_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗