Pytowski, L. | bioRxiv Science

bioRxiv2019

A novel technique for mapping viscosity in discrete subcellular locations with a BODIPY based fluorescent probe

Numerous cellular processes, including enzyme behaviour, signalling, and protein folding and transport are highly influenced by the local microviscosity environment within living cells. Molecular rotors are fluorescent molecules that respond to the viscosity of their environment through changes in both the intensity and lifetime of their fluorescence. We have synthesised a novel benzyl-guanine derivatized boron-dipyrromethene (BODIPY) molecular rotor that is a substrate for the SNAP-tag targeting system (named BG-BODIPY), allowing us to target the rotor to discrete locations within the living cell. We demonstrate that BG-BODIPY reports viscosity, and that this can be measured either through fluorescence lifetime or intensity ratiometric measurements. The relative microviscosities within the ER, Golgi, mitochondrial matrix, peroxisomes, lysosomes, cytoplasm, and nucleoplasm were significantly different. Additionally, this approach permitted fluorescence lifetime imaging microscopy (FLIM) to determine the absolute viscosity within both mitochondria and stress granules, showcasing BG-BODIPYs usefulness in studying both membrane-bound and membraneless organelles. These results highlight targeted BG-BODIPYs broad usefulness for making measurements of cellular viscosity both with FLIM and conventional ratiometric confocal microscopy, the latter option greatly extending the accessibility of the technique although limited to relative meassurements. Statement of SignificanceLocal viscosity affects molecular behaviour from diffusion and conformational changes to enzyme kinetics and has important implications for cell and tissue function. Mechanical methods for measurement of viscosity average over large volumes and long times and are thus unsuitable for rapid changes on small scales that are biologically relevant. This paper reports a novel optical fluorescence method using genome edited cells to deliver a viscosity reporter to tightly defined locations inside living cells, from which non-destructive organelle-specific measurements can be repeatedly made. The local viscosity of seven separate organelles in living cultured human cells is shown for the first time, together with the viscosity behaviour of a membraneless organelle as it is induced in cells by stress.

cell biology↗

bioRxiv2016

Host Vesicle Fusion Proteins VAPB, Rab11b and Rab18 Contribute to HSV-1 Infectivity by Facilitating Egress through the Nuclear Membrane

The herpesvirus process of primary envelopment and de-envelopment as viral particles exit the nucleus has been for many years one of the least understood steps in the virus life cycle. Though viral proteins such as pUL31, pUL34, pUS3 and others are clearly important, these are likely insufficient for efficient fusion with the nuclear membrane. We postulated that host nuclear membrane proteins involved in virus nuclear egress would move from the inner to outer nuclear membranes due to membrane fusion events in primary envelopment and de-envelopment and then diffuse into the endoplasmic reticulum. Membrane fractions were prepared enriched in the nuclear envelope or the endoplasmic reticulum with and without HSV-1 infection and analyzed by mass spectrometry, revealing several vesicle fusion proteins as candidates in the viral nuclear egress pathway. Knockdown of three of these, VAPB, Rab11b, and Rab18, significantly reduced titers of released virus while yielding nuclear accumulation of encapsidated particles. Antibody staining revealed that VAPB visually accumulates in the inner nuclear membrane during HSV-1 infection. VAPB also co-localizes at early time points with the viral pUL34 protein known to be involved in nuclear egress. Most strikingly, VAPB was also observed on HSV-1 virus particles by immunogold labelling electron microscopy. Thus, these data reveal several new host cell vesicle fusion proteins involved in viral nuclear egress.\n\nAuthor SummaryHuman herpesviruses are associated with common human diseases such as chicken pox, shingles and mononucleosis and infect a wide range of animals making them economically important pathogens for livestock. Herpes simplex virus 1 (HSV-1) is most commonly associated with cold sores, but is also the leading cause of blindness by infection in the Western world. All herpesviruses share many aspects of infection. As large nuclear replicating dsDNA viruses with capsid sizes too large to use the nuclear pores to exit the nucleus, they have evolved a complex mechanism for envelopment and de-envelopment of primary herpesvirus particles, but this critical step in the virus lifecycle remains poorly understood. We have identified several host cell vesicle fusion proteins, VAPB, Rab11b and Rab18 that appear to contribute to this step in the HSV-1 life cycle. VAPB accumulates at the nuclear envelope with the HSV-1 pUL34 protein important for viral nuclear egress. Knockdown of any of these vesicle fusion proteins reduces viral titers, further arguing that they are important for nuclear egress. As there appears to be a specific subset of vesicle fusion proteins involved in viral egress, they could possibly represent novel targets for therapeutic interventions.

cell biology↗