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Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli.

Bakshi, S and Bratton, BP and Weisshaar, JC (2011) Subdiffraction-limit study of Kaede diffusion and spatial distribution in live Escherichia coli. Biophys J, 101. pp. 2535-2544.

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Abstract

Photoactivation localization microscopy (PALM) is used to study the spatial distribution and diffusion of single copies of the protein Kaede in the cytoplasm of live Escherichia coli under moderate growth conditions (67 min doubling time). The spatial distribution of Kaede is uniform within the cytoplasm. The cytoplasmic radius of 380 ± 30 nm varies little from cell to cell. Single-particle tracking using 4 ms exposure times reveals negatively curved plots of mean-square displacement versus time. A detailed comparison with Monte Carlo simulations in a spherocylindrical volume shows that the curvature can be quantitatively understood in terms of free diffusion within a confining volume. The mean diffusion coefficient across cells is <D(Kaede)> = 7.3 ± 1.1 μm(2)·s(-1), consistent with a homotetrameric form of Kaede. The distribution of squared displacements along the long axis for individual Kaede molecules is consistent with homogeneous diffusion. However, for longer cells, a spatial map of one-step estimates of the diffusion coefficient along x suggests that diffusion is ∼20-40% faster within nucleoids than in the ribosome-rich region lying between nucleoid lobes at the cell mid-plane. Fluorescence recovery after photobleaching yielded <D(FRAP)> = 8.3 ± 1.6 μm(2)·s(-1), in agreement with the single-particle tracking results.

Item Type: Article
Uncontrolled Keywords: Cephalexin Diffusion Escherichia coli Fluorescence Recovery After Photobleaching Luminescent Proteins Microbial Viability Microscopy, Fluorescence Protein Transport Ribosomes Time Factors
Subjects: UNSPECIFIED
Divisions: Div F > Control
Depositing User: Cron Job
Date Deposited: 21 May 2020 02:13
Last Modified: 09 Sep 2021 00:38
DOI: 10.1016/j.bpj.2011.10.013