Biology subjects

Shrestha, D.

Publications and source records attributed to Shrestha, D..

3 recordsLinked to original sources

bioRxiv2018

Lipid composition but not curvature is a determinant of a low molecular mobility within HIV-1 lipid envelope

Human Immunodeficiency Virus type-1 (HIV-1) acquires its lipid membrane from the plasma membrane of the infected cell from where it buds out. Previous studies have shown that the HIV-1 envelope is a very low mobility environment with the diffusion of incorporated proteins two orders of magnitude slower than in plasma membrane. One of the reasons for this difference is thought to be due to HIV-1 membrane composition that is characterised by a high degree of rigidity and lipid packing. To further refine the model of the molecular mobility on HIV-1 surface, we here investigated the relative importance of membrane composition and curvature in Large Unilamellar Vesicles of different composition and size (0.2-1 m) by super-resolution STED microscopy-based fluorescence correlation spectroscopy (STED-FCS) analysis. We find that lipid composition and its rigidity but not membrane curvature play an important role in the decreased molecular mobility on vesicle surface thus confirming that this factor is an essential determinant of HIV-1 low surface mobility. Our results provide further insight into the dynamic properties of the surface of individual HIV-1 particles.

biophysics↗

bioRxiv2017

Complementary studies of lipid membrane dynamics using iSCAT and STED microscopy

Observation techniques with high spatial and temporal resolution, such as single-particle tracking (SPT) based on interferometric Scattering (iSCAT) microscopy, and fluorescence correlation spectroscopy applied on a super-resolution STED microscope (STED-FCS), have revealed new insights of the molecular organization of membranes. While delivering complementary information, there are still distinct differences between these techniques, most prominently the use of fluorescent dye-tagged probes for STED-FCS and a need for larger scattering gold nanoparticle tags for iSCAT. In this work we have used lipid analogues tagged with a hybrid fluorescent tag - gold nanoparticle construct, to directly compare the results from STED-FCS and iSCAT measurements of phospholipid diffusion on a homogeneous Supported Lipid Bilayer (SLB). These comparative measurements showed that while the mode of diffusion remained free, at least at the spatial (>40 nm) and temporal (50 [≤] t [≤] 100 ms) scales probed, the diffussion coefficient was reduced by 20- to 60-fold when tagging with 20 and 40 nm large gold particles as compared to when using dye-tagged lipid analogues. These FCS measurements of hybrid fluorescent tag - gold nanoparticle labeled lipids also revealed that commercially supplied streptavidin-coated gold nanoparticles contain large quantities of free streptavidin. Finally, the values of apparent diffusion coefficients obtained by STED-FCS and iSCAT differed by a factor of 2-3 across the techniques, while relative differences in mobility between different species of lipid analogues considered were identical in both approaches. In conclusion, our experiments reveal that large and potentially crosslinking scattering tags introduce a significant slow-down in diffusion on SLBs but no additional bias, and our labeling approach creates a new way of exploiting complementary information from STED-FCS and iSCAT measurements.

biophysics↗

bioRxiv2017

Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

Scanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

biophysics↗