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Exciting Insights into Cell Growth

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THE QUESTION

How do cells grow?

As a cell increases in size during progression of the cell cycle, how is new material added to the cell wall or the plasma membrane? Does growth occur evenly across the cell or are there specific zones where new components are deposited? Is there a pattern to addition? How is turgor pressure maintained? These are fundamental questions about the biology of a cell—as fundamental as DNA replication— but many of the molecular details remain unclear.1,2

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An interdisciplinary team of researchers at the University of Sheffield, including physicist Ashley Cadby, are trying to change this situation—

Specifically, they are shedding light on how gram-negative bacteria add peptidoglycan to their cell wall.2 The 40 nm resolution they achieved using STORM revealed surprising new details of this process, forcing Turner and colleagues to reshape their models of cell wall growth.

Above: New cell wall growth on S. aureus, using vancomycin labeled with Alexa Fluor 532, 60x magnification and 107 nm pixel.
Video courtesy of Ashley Cadby, EPMM Group, University of Sheffield.

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Read the Paper (login may be required)
Turner, R. D., Hurd, A. F., Cadby, A., Hobbs, J. K. & Foster, S. J. Cell wall elongation mode in gram-negative bacteria is determined by peptidoglycan architecture. Nat. Commun. 4, 1496 (2013).
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Super-resolution with CMOS
Super-resolution microscopy has traditionally been done using EM-CCD cameras. See how Huang, et al., used a CMOS camera for video-rate super-resolution microscopy.
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Stochastic Optical Reconstruction Microscopy

Excited single molecules are read out from random locations.
Excited fluorophores are switched off by switching to a different excited state, not necessarily the bleached state (“switched off” state is the same as the unexcited ground state). Resolution at < 40 nm

STORM Conditions

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Diode Laser:
100 mW, 532 nm diode laser (Laser 2000)
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Microscope:
Olympus IX71 inverted optical microscope
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Objectives:
PlanApochromat 60x oil immersion objective, 1.4 NA
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Camera:
ImagEM camera (Hamamatsu)
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Filter:
552-nm longpass dichroic filter(Semrock FF552-DI02)
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Filter:
565(24)-nm bandpass emission filter (Semrock Brightline 565/24)
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Image Expander:
35mm and 100mm lenes
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Focus:
1 TB Focus adjusted via piezoelectric motor (Physik Instrumente)
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Previous models of bacterial cell wall growth include (A) helical deposition3 versus (B) an irregular, patchy mosaic of new growth sites.4,5

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(A) Helical deposition

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(B) Diffuse Deposition

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THE BARRIERS

How do you visualize processes occurring in the low nanometer scale, but in the context of a living cell that’s >1000 times larger?

A big technical hurdle in studying bacterial cell wall synthesis has been the scales that these events occur at. Another example to think about: If peptidoglycan equals one person, then E. coli equals one mile or 2 kilometers. In addition, earlier fluorescence microscopy techniques have been limited by the light diffraction barrier of 200 nm. Read more about breaking the light diffraction barrier in Exciting Advances Push the Limits of Visualization.

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Above: Wide fields-of-view using the ORCA-Flash4.0. FluoCells imaged with an S Plan Fluor 100x objective. Image from Hamamatsu Cameras.

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THE SOLUTION

Turner, et al,2 used fluorescently labeled vancomycin to identify regions of peptidoglycan insertion—the antibiotic vancomycin binds to the D-ala-D-ala motif present in unmodified, and therefore nascent, material.

To ensure that the labeled vancomycin entered the cell, they developed a method to fix and treat E. coli cells to allow vancomycin binding.2 When Turner, et al,2 imaged their samples—the gram-negative bacteria E. coli and C. crescentus—using deconvolution fluorescence microscopy, they found nascent peptidoglycan localizing to division septa, as expected, high labeling at the poles, which was unexpected, and diffuse labeling throughout the body of the cell.

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To better understand what was happening at the nanometer scale, they turned to STORM, achieving resolution between 35 and 42 nm. What had been diffuse staining throughout the body of the cell resolved into a peppering of distinct foci randomly distributed across the surface of the cell and ranging from single motifs to 50 nm clusters. This arrangement is in surprising contrast to the helical path traveled by the cell elongation machinery. Together with the results from atomic force microscopy (AFM) studies, Turner, et al,2 propose a new model for cell wall growth, where peptidoglycan insertion happens in the least dense regions of the cell wall.

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Above: STORM images of E. coli (MG1655) sacculi showing multiple, distinct foci of insertion using fluorescent vancomycin (Alexa Fluor 532). Courtesy of Ashley Cadby, EPMM Group, University of Sheffield.

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50 Frames Per Second

Turner, et al,2 turned to super-resolution microscopy to image events in the low nanometer regime. Hamamatsu’s ImagEM camera was part of the STORM setup, capturing the 35-42 nm -resolution images at 50 frames per second. Learn more about ImagEM.

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What can camera specs tell you?
For those new to microscopy and imaging, looking through a camera specifications sheet can be like trying to decipher Greek. To learn what all the terms mean, read “Dissecting camera specifications: a field guide for biologists.”
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Widefield imaging best practices
Whether you’re imaging bacteria or eukaryotic cells with an ImagEM, CCD, or an sCMOS camera, there are some basic steps you can take to ensure your images reflect what’s happening in the micrometer and nanometer scale— learn more.
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THE POSSIBILITIES

Turner, et al,2 used super-resolution microscopy to see nanometer-scale events in the bacterial cell wall.

This powerful technique—really a suite of techniques—is giving a growing number of researchers the ability to see further into biology than they ever have before. From the basic biology of HIV to experiments into how myosin-1A binds to the membrane, researchers across the globe are using Hamamatsu’s ImagEM camera for studies at the micro- and nano-scale. See how in the selected papers below2, available at PubMed Central.

LEDGINs inhibit late stage HIV-1 replication by modulating integrase multimerization in the virions

Belete Ayele Desimmie, Rik Schrijvers, Jonas Demeulemeester, Doortje Borrenberghs, Caroline Weydert, Wannes Thys, Sofie Vets, Barbara Van Remoortel, Johan Hofkens, Jan De Rijck, Jelle Hendrix, Norbert Bannert, Rik Gijsbers, Frauke Christ, and Zeger Deby.

RETROVIROLOGY. 2013; 10: 57. PMCID: PMC3671127

Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain

Jessica N. Mazerik and Matthew J. Tysk

J BIOL CHEM. 2012 APRIL 13; 287(16): 13104–13115. PMCID: PMC3339983

Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1 Interaction

Martin Lehmann, Susana Rocha, Bastien Mangeat, Fabien Blanchet, Hiroshi Uji-i, Johan Hofkens, and Vincent Piguet

PLOS PATHOG. 2011 DECEMBER; 7(12): E1002456. PMCID: PMC3240612

REFERENCES

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  2. 02. Turner, R. D., Hurd, A. F., Cadby, A., Hobbs, J. K. & Foster, S. J. Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture. Nat. Commun. 4, 1496 (2013).
  3. 03. Varma, A., Pedro, M. A. de & Young, K. D. FtsZ Directs a Second Mode of Peptidoglycan Synthesis in Escherichia coli. J. Bacteriol. 189, 5692–5704 (2007).
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