Pleckstrin homology domain diffusion in Dictyostelium cytoplasm studied using fluorescence correlation spectroscopy

A. Ruchira, M.A. Hink, L. Bosgraaf, P.J.M. van Haastert, A.J.W.G. Visser

Research output: Contribution to journalArticleAcademicpeer-review

21 Citations (Scopus)

Abstract

The translocation of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to the plasma membrane plays an important role in the chemotaxis mechanism of Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein (GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium cells has been studied using fluorescence correlation spectroscopy to gain a better understanding of the functioning of the domains and to assess the effect of initiation of chemotaxis on these domains in the cell. PH2-GFP was homogeneously distributed in vegetative as well as chemotaxing cells, whereas PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were very similar; however, PH10-GFP exhibited slower diffusion. Photon counting histogram statistics show that this slow diffusion was not due to aggregation. Diffusion of the three PH domains was affected to similar extents by intracellular heterogeneities in vegetative as well as chemotaxing cells. From the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP and PH-CRAC-GFP remained unchanged.
Original languageEnglish
Pages (from-to)10013-10019
JournalJournal of Biological Chemistry
Volume279
Issue number11
DOIs
Publication statusPublished - 2004

Keywords

  • photon-counting histogram
  • fluctuation spectroscopy
  • molecular-dynamics
  • fusion proteins
  • ph domains
  • cells
  • binding
  • level

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