PRIM: Proximity imaging of green fluorescent protein-tagged polypeptides

We report a serendipitous discovery that extends the impressive catalog of reporter functions performed by green fluorescent protein (GFP) or its derivatives. When two GFP molecules are brought into proximity, changes in the relative intensities of green fluorescence emitted upon excitation at 395 vs. 475 nm result. These spectral changes provide a sensitive ratiometric index of the extent of self-association that can be exploited to quantitatively image homo-oligomerization or clustering processes of GFP-tagged proteins in vivo. The method, which we term proximity imaging (PRIM), complements fluorescence resonance energy transfer between a blue fluorescent protein donor and a GFP acceptor, a powerful method for imaging proximity relationships between different proteins. However, unlike fluorescence resonance energy transfer (which is a spectral interaction), PRIM depends on direct contact between two GFP modules, which can lead to structural perturbations and concomitant spectral changes within a module. Moreover, the precise spatial arrangement of the GFP molecules within a given dimer determines the magnitude and direction of the spectral change. We have used PRIM to detect FK1012-induced dimerization of GFP fused to FK506-binding protein and clustering of glycosylphosphatidylinositol-anchored GFP at cell surfaces.

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.

Identification of Two Type V Myosins in Fission Yeast, One of Which Functions in Polarized Cell Growth and Moves Rapidly in the Cell

We characterized the novel Schizosaccharomyces pombe genes myo4+ and myo5+, both of which encode myosin-V heavy chains. Disruption of myo4 caused a defect in cell growth and led to an abnormal accumulation of secretory vesicles throughout the cytoplasm. The mutant cells were rounder than normal, although the sites for cell polarization were still established. Elongation of the cell ends and completion of septation required more time than in wild-type cells, indicating that Myo4 functions in polarized growth both at the cell ends and during septation. Consistent with this conclusion, Myo4 was localized around the growing cell ends, the medial F-actin ring, and the septum as a cluster of dot structures. In living cells, the dots of green fluorescent protein-tagged Myo4 moved rapidly around these regions. The localization and movement of Myo4 were dependent on both F-actin cables and its motor activity but seemed to be independent of microtubules. Moreover, the motor activity of Myo4 was essential for its function. These results suggest that Myo4 is involved in polarized cell growth by moving with a secretory vesicle along the F-actin cables around the sites for polarization. In contrast, the phenotype of myo5 null cells was indistinguishable from that of wild-type cells. This and other data suggest that Myo5 has a role distinct from that of Myo4.