In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography
- California Institute of Technology, United States
- Leiden University, United States
- University of Kentucky, United States
- University of Southern California, United States
- University of Pennsylvania, United States
- Imperial College London, United Kingdom
- Hampshire College, United States
- Minnesota State University, United States
- University of Wisconsin, United States
- Leiden University, Netherlands
Abstract
The ability to produce outer membrane projections in the form of tubular membrane extensions (MEs) and membrane vesicles (MVs) is a widespread phenomenon among diderm bacteria. Despite this, our knowledge of the ultrastructure of these extensions and their associated protein complexes remains limited. Here, we surveyed the ultrastructure and formation of MEs and MVs, and their associated protein complexes, in tens of thousands of electron cryo-tomograms of ~ 90 bacterial species that we have collected for various projects over the past 15 years (Jensen lab database), in addition to data generated in the Briegel lab. We identified outer MEs and MVs in 13 diderm bacterial species and classified several major ultrastructures: 1) tubes with a uniform diameter (with or without an internal scaffold), 2) tubes with irregular diameter, 3) tubes with a vesicular dilation at their tip, 4) pearling tubes, 5) connected chains of vesicles (with or without neck-like connectors), 6) budding vesicles and nanopods. We also identified several protein complexes associated with these MEs and MVs which were distributed either randomly or exclusively at the tip. These complexes include a secretin-like structure and a novel crown-shaped structure observed primarily in vesicles from lysed cells. In total, this work helps to characterize the diversity of bacterial membrane projections and lays the groundwork for future research in this field.
Data availability
All data generated or analysed during this study are included in the manuscript and supporting files and movies.
Article and author information
Author details
Lauren Ann Metskas
Catherine M Oikonomou
Funding
Foundation for the National Institutes of Health (R35 GM122588)
- Grant J Jensen
Baxter Postdoctoral fellowship
- Mohammed Kaplan
Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO OCENW.GROOT.2019.063)
- Mohammed Kaplan
National Institutes of Health (P20 GM130456)
- Carrie Shaffer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Copyright
© 2021, Kaplan et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
In situ imaging of bacterial outer membrane projections and associated protein complexes using electron cryo-tomography
eLife 10:e73099.
https://doi.org/10.7554/eLife.73099
Further reading
-
- Microbiology and Infectious Disease
A combination of imaging techniques reveals how herpes simplex virus type 1 assembles within infected cells, highlighting the roles of essential viral proteins in viral assembly and exit.
-
- Microbiology and Infectious Disease
Virion Infectivity Factor (Vif) of the Human Immunodeficiency Virus type 1 (HIV-1) targets and degrades cellular APOBEC3 proteins, key regulators of intrinsic and innate antiretroviral immune responses, thereby facilitating HIV-1 infection. While Vif’s role in degrading APOBEC3G is well-studied, Vif is also known to cause cell cycle arrest, but the detailed nature of Vif’s effects on the cell cycle has yet to be delineated. In this study, we employed high-temporal resolution single-cell live imaging and super-resolution microscopy to monitor individual cells during Vif-induced cell cycle arrest. Our findings reveal that Vif does not affect the G2/M boundary as previously thought. Instead, Vif triggers a unique and robust pseudo-metaphase arrest, distinct from the mild prometaphase arrest induced by Vpr. During this arrest, chromosomes align properly and form the metaphase plate, but later lose alignment, resulting in polar chromosomes. Notably, Vif, unlike Vpr, significantly reduces the levels of both Protein Phosphatase 1 (PP1) and 2 A (PP2A) at kinetochores, which regulate chromosome-microtubule interactions. These results unveil a novel role for Vif in kinetochore regulation that governs the spatial organization of chromosomes during mitosis.
