Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, Mitochondrial Dynamics, and Cell Growth

MicroRNA (miRNA) has emerged as an important gene-regulator that shows great potential in gene therapy because of its unique roles in gene-regulation. However, the knowledge on their function and transportation in vivo is still lacking, and there are limited obvious evidences to define intracellular transportation of miRNA. In this study, the dynamics of exogenous miR-21 transfected into HeLa cells was traced by live-cell microscopy. Their transportation at key time points was recorded and dynamic properties were analyzed by single particle tracking (SPT) and mean square displacement (MSD) calculation. Results showed that the exogenous miRNAs bounded to cells quickly and went through lysosome into cytosol, where they were subsequently recruited into p-body. They finally were degraded, otherwise went back to cytosol in some way. Long time observation and analysis of motion mode showed that the miRNAs were confined in a small region and their motion modes were flexible in different intracellular microenvironment after entering the cells.

Download Full-text

Direct lysosome-based autophagy of lipid droplets in hepatocytes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2011442117 ◽

2020 ◽

Vol 117 (51) ◽

pp. 32443-32452

Author(s):

Ryan J. Schulze ◽

Eugene W. Krueger ◽

Shaun G. Weller ◽

Katherine M. Johnson ◽

Carol A. Casey ◽

...

Keyword(s):

Mammalian Cells ◽

Lipid Droplets ◽

Critical Role ◽

Specific Protein ◽

Lipid Homeostasis ◽

Dynamic Interactions ◽

Double Membrane ◽

Live Cell Microscopy ◽

Cell Microscopy

Hepatocytes metabolize energy-rich cytoplasmic lipid droplets (LDs) in the lysosome-directed process of autophagy. An organelle-selective form of this process (macrolipophagy) results in the engulfment of LDs within double-membrane delimited structures (autophagosomes) before lysosomal fusion. Whether this is an exclusive autophagic mechanism used by hepatocytes to catabolize LDs is unclear. It is also unknown whether lysosomes alone might be sufficient to mediate LD turnover in the absence of an autophagosomal intermediate. We performed live-cell microscopy of hepatocytes to monitor the dynamic interactions between lysosomes and LDs in real-time. We additionally used a fluorescent variant of the LD-specific protein (PLIN2) that exhibits altered fluorescence in response to LD interactions with the lysosome. We find that mammalian lysosomes and LDs undergo interactions during which proteins and lipids can be transferred from LDs directly into lysosomes. Electron microscopy (EM) of primary hepatocytes or hepatocyte-derived cell lines supports the existence of these interactions. It reveals a dramatic process whereby the lipid contents of the LD can be “extruded” directly into the lysosomal lumen under nutrient-limited conditions. Significantly, these interactions are not affected by perturbations to crucial components of the canonical macroautophagy machinery and can occur in the absence of double-membrane lipoautophagosomes. These findings implicate the existence of an autophagic mechanism used by mammalian cells for the direct transfer of LD components into the lysosome for breakdown. This process further emphasizes the critical role of lysosomes in hepatic LD catabolism and provides insights into the mechanisms underlying lipid homeostasis in the liver.

Download Full-text

Visualization of Chromatin Dynamics by Live Cell Microscopy Using CRISPR/Cas9 Gene Editing and ANCHOR Labeling

Methods in Molecular Biology - Capturing Chromosome Conformation ◽

10.1007/978-1-0716-0664-3_11 ◽

2020 ◽

pp. 197-212

Author(s):

Ezio T. Fok ◽

Stephanie Fanucchi ◽

Kerstin Bystricky ◽

Musa M. Mhlanga

Keyword(s):

Gene Editing ◽

Live Cell ◽

Chromatin Dynamics ◽

Live Cell Microscopy ◽

Cell Microscopy

Download Full-text

Investigating the Role of F-Actin in Human Immunodeficiency Virus Assembly by Live-Cell Microscopy

Journal of Virology ◽

10.1128/jvi.00431-14 ◽

2014 ◽

Vol 88 (14) ◽

pp. 7904-7914 ◽

Cited By ~ 14

Author(s):

Sheikh Abdul Rahman ◽

Peter Koch ◽

Julian Weichsel ◽

William J. Godinez ◽

Ulrich Schwarz ◽

...

Keyword(s):

Plasma Membrane ◽

Actin Filaments ◽

Virus Assembly ◽

Live Cell ◽

Viral Budding ◽

Immunodeficiency Virus ◽

Live Cell Microscopy ◽

Gag Assembly ◽

Hiv 1

ABSTRACTHuman immunodeficiency virus type 1 (HIV-1) particles assemble at the plasma membrane, which is lined by a dense network of filamentous actin (F-actin). Large amounts of actin have been detected in HIV-1 virions, proposed to be incorporated by interactions with the nucleocapsid domain of the viral polyprotein Gag. Previous studies addressing the role of F-actin in HIV-1 particle formation using F-actin-interfering drugs did not yield consistent results. Filamentous structures pointing toward nascent HIV-1 budding sites, detected by cryo-electron tomography and atomic force microscopy, prompted us to revisit the role of F-actin in HIV-1 assembly by live-cell microscopy. HeLa cells coexpressing HIV-1 carrying fluorescently labeled Gag and a labeled F-actin-binding peptide were imaged by live-cell total internal reflection fluorescence microscopy (TIR-FM). Computational analysis of image series did not reveal characteristic patterns of F-actin in the vicinity of viral budding sites. Furthermore, no transient recruitment of F-actin during bud formation was detected by monitoring fluorescence intensity changes at nascent HIV-1 assembly sites. The chosen approach allowed us to measure the effect of F-actin-interfering drugs on the assembly of individual virions in parallel with monitoring changes in the F-actin network of the respective cell. Treatment of cells with latrunculin did not affect the efficiency and dynamics of Gag assembly under conditions resulting in the disruption of F-actin filaments. Normal assembly rates were also observed upon transient stabilization of F-actin by short-term treatment with jasplakinolide. Taken together, these findings indicate that actin filament dynamics are dispensable for HIV-1 Gag assembly at the plasma membrane of HeLa cells.IMPORTANCEHIV-1 particles assemble at the plasma membrane of virus-producing cells. This membrane is lined by a dense network of actin filaments that might either present a physical obstacle to the formation of virus particles or generate force promoting the assembly process. Drug-mediated interference with the actin cytoskeleton showed different results for the formation of retroviral particles in different studies, likely due to general effects on the cell upon prolonged drug treatment. Here, we characterized the effect of actin-interfering compounds on the HIV-1 assembly process by direct observation of virus formation in live cells, which allowed us to measure assembly rate constants directly upon drug addition. Virus assembly proceeded with normal rates when actin filaments were either disrupted or stabilized. Taken together with the absence of characteristic actin filament patterns at viral budding sites in our analyses, this indicates that the actin network is dispensable for HIV-1 assembly.

Download Full-text