Characterization of Protein Dynamics in Asymmetric Cell Division by Scanning Fluorescence Correlation Spectroscopy

Biophysics Group, Biotechnologisches Zentrum, Technische Universität Dresden, Dresden, Germany.
Biophysical Journal (Impact Factor: 3.97). 10/2008; 95(11):5476-86. DOI: 10.1529/biophysj.108.135152
Source: PubMed


The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

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Available from: Alireza Mashaghi, Apr 14, 2014

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    ABSTRACT: Two-photon fluorescence imaging of proteins labelled with GFP or its analogues provides information on the localization of the molecules in cells and tissues, and their redistribution on timescales as short as milliseconds. Fluorescence correlation spectroscopy (FCS) analyzes fluctuations of the fluorescence signal in order to yield information about the motion of the molecules on timescales considerably shorter than those accessible with imaging, allowing the determination of diffusion coefficients, estimation of aggregate size, molecular concentrations, etc., i. e., parameters that can be difficult to determine with imaging alone. Scanning FCS (sFCS) is a modification of FCS that provides information about molecular dynamics and type of motion, which is too slow for standard FCS, and not resolvable with imaging. We have applied two-photon imaging, FCS and sFCS to study the localization and redistribution of GFP-labelled proteins involved in the asymmetric first division of C. elegans embryos. While the distribution of the investigated proteins in the cytoplasm is homogeneous on the scale limited by the optical resolution and their fast motion can be well characterized with conventional FCS, the proteins localized in the cortex exhibit patterns evolving on the ms-s temporal scale. We use sFCS and explore the applicability of spatial correlation analysis (image correlation, STICS) to the qualitative and quantitative description of the dynamics of the cortex-localized proteins.
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