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Chapter 3 : The Dynamic Architecture of the Cell

Authors: Marc D. Sharp, Kit Pogliano
Content Type: Monograph
Publication Year: 2002

Category: Bacterial Pathogenesis

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Abstract:

This chapter discusses a few observations that have revealed the bacterial cell to be both dynamic and exquisitely organized, illustrates some of the techniques that have allowed these studies, and introduces a few new technologies that promise to provide an even more detailed and quantitative view of individual bacterial cells. In eukaryotic cells, directed movement of many macromolecules requires the cytoskeleton and associated motor proteins, which are lacking from bacterial cells. The large number of proteins found within the sporulation septum suggests that the septal membrane domain is functionally and structurally distinct from the remainder of the cytoplasmic membrane. While it is very difficult or impossible to visualize bacterial septa reproducibly using phase-contrast or Nomarski optics, fluorescent membrane stains are able to detect even incompletely synthesized septa, and they allow the precise measurement of cell length for studies of the bacterial cell cycle. Some of the most exciting new optical methods move well beyond the structural characterization of the bacterial cells to allow the quantitative biochemical analysis of individual cells and molecules. For example, fluorescence resonance energy transfer (FRET) between two different fluorophores (such as the spectral variants of GFP) allows measurements of the distance between two molecules on a nanometer scale. Few organisms are as amenable to genetic manipulation and cell biological studies as , and there is a wealth of molecular and biochemical information about pathways for morphogenesis, signal transduction, and the cell cycle.

Citation: Sharp M, Pogliano K. 2002. The Dynamic Architecture of the Cell, p 15-20. In Sonenshein A, Losick R, Hoch J (ed), and Its Closest Relatives. ASM Press, Washington, DC. doi: 10.1128/9781555817992.ch3
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The Dynamic Architecture of the Bacillus Cell
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Citation: Sharp M, Pogliano K. 2002. The Dynamic Architecture of the Cell, p 15-20. In Sonenshein A, Losick R, Hoch J (ed), and Its Closest Relatives. ASM Press, Washington, DC. doi: 10.1128/9781555817992.ch3
/content/10.1128/9781555817992.chap3.fig3-1
FIGURE 1

The complicated architecture of the forespore chromosome revealed by DAPI staining, deconvolution, and optical sectioning microscopy. Sporangia were fixed ( and stained with DAPI 4 h after the initiation of sporulation. By this time, the forespore chromosome has become highly condensed through the action of the SASP proteins, which force the chromosomes into a ring structure. Prior to deconvolution (A), the forespore chromosomes appear as highly condensed rings or lines (arrows 1 to 3), whereas the mother cell and vegetative chromosomes are more diffuse. Following deconvolution (B), the morphology of the forespore chromosome is more clear; however, a great deal of information is lost if the image is overadjusted to increase image contrast (C). A similar effect will be seen if the camera or film is saturated during image acquisition. The lower panels show enlarged optical sections through the forespore chromosomes indicated by numbered arrows in panels A to C. The numbers in the upper left corner correspond to the arrows in panels A to C, and the numbers in the upper right corner indicate the number of microns above or below the focal plane at which each image was taken. Forespore chromosome no. 1 is slightly folded, while forespore chromosome no. 2 appears to be a twisted figure eight, with one loop perpendicular to the focal plane, and forespore chromosome no. 3 has a more complicated topology. Even prior to deconvolution, there is a notable difference in sections spaced by just 0.18 μm, demonstrating the importance of fine focus control. Bars, 0.75 μm.

10.1128/9781555817992/fig3-1_thmb.gif
10.1128/9781555817992/fig3-1.gif
Image of FIGURE 1
FIGURE 1

The complicated architecture of the forespore chromosome revealed by DAPI staining, deconvolution, and optical sectioning microscopy. Sporangia were fixed ( and stained with DAPI 4 h after the initiation of sporulation. By this time, the forespore chromosome has become highly condensed through the action of the SASP proteins, which force the chromosomes into a ring structure. Prior to deconvolution (A), the forespore chromosomes appear as highly condensed rings or lines (arrows 1 to 3), whereas the mother cell and vegetative chromosomes are more diffuse. Following deconvolution (B), the morphology of the forespore chromosome is more clear; however, a great deal of information is lost if the image is overadjusted to increase image contrast (C). A similar effect will be seen if the camera or film is saturated during image acquisition. The lower panels show enlarged optical sections through the forespore chromosomes indicated by numbered arrows in panels A to C. The numbers in the upper left corner correspond to the arrows in panels A to C, and the numbers in the upper right corner indicate the number of microns above or below the focal plane at which each image was taken. Forespore chromosome no. 1 is slightly folded, while forespore chromosome no. 2 appears to be a twisted figure eight, with one loop perpendicular to the focal plane, and forespore chromosome no. 3 has a more complicated topology. Even prior to deconvolution, there is a notable difference in sections spaced by just 0.18 μm, demonstrating the importance of fine focus control. Bars, 0.75 μm.

Citation: Sharp M, Pogliano K. 2002. The Dynamic Architecture of the Cell, p 15-20. In Sonenshein A, Losick R, Hoch J (ed), and Its Closest Relatives. ASM Press, Washington, DC. doi: 10.1128/9781555817992.ch3
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/content/10.1128/9781555817992.chap3.fig3-2
FIGURE 2

Three-dimensional reconstruction of a forespore chromosome ring. Images were collected at focal planes spaced by 0.06 μm through a forespore chromosome ring lying nearly perpendicular to the focal plane. (A) Panels i to iii show three of the 30 optical sections collected for this reconstruction. Panel iv is an overlay of panels i and iii that begins to show the three-dimensional structure of the chromosome ring. (B) Panels show three rotations of the chromosome ring after reconstruction. The 0° rotation closely corresponds to the image shown in panel iv, while the -45° and -90° rotations clearly show that the reconstructed object is, indeed, a ring. Bar, 0.5 μm

10.1128/9781555817992/fig3-2_thmb.gif
10.1128/9781555817992/fig3-2.gif
Image of FIGURE 2
FIGURE 2

Three-dimensional reconstruction of a forespore chromosome ring. Images were collected at focal planes spaced by 0.06 μm through a forespore chromosome ring lying nearly perpendicular to the focal plane. (A) Panels i to iii show three of the 30 optical sections collected for this reconstruction. Panel iv is an overlay of panels i and iii that begins to show the three-dimensional structure of the chromosome ring. (B) Panels show three rotations of the chromosome ring after reconstruction. The 0° rotation closely corresponds to the image shown in panel iv, while the -45° and -90° rotations clearly show that the reconstructed object is, indeed, a ring. Bar, 0.5 μm

Citation: Sharp M, Pogliano K. 2002. The Dynamic Architecture of the Cell, p 15-20. In Sonenshein A, Losick R, Hoch J (ed), and Its Closest Relatives. ASM Press, Washington, DC. doi: 10.1128/9781555817992.ch3
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