Dictyostelium Chemotaxis studied with fluorescence fluctuation spectroscopy

The movement of cells in the direction of a chemical gradient, also known as chemotaxis, is a vital biological process. During chemotaxis, minute extracellular signals are translated into complex cellular responses such as change in morphology and motility. To understand the chemotaxis mechanism at a molecular level it is important to obtain detailed information about the localization and dynamics of the signaling processes. For the study of chemotaxis the organism Dictyostelium</span><span lang=EN-US style='mso-bidi-font-size:4.0pt;line-height:130%;color:black; mso-ansi-language:EN-US'>discoideum</span><span lang=EN-US style='mso-bidi-font-size:4.0pt;line-height:130%;color:black;mso-ansi-language: EN-US'>is used as a model organism.</span><span lang=EN-US style='mso-ansi-language: EN-US'>Dictyostelium shows remarkable similarity in chemotaxis to mammalian cells and has the advantage that it is genetically manageable and easily accessible for biochemical and biological studies. In this thesis, information about various regulatory molecules involved in the chemotaxis mechanism of Dictyostelium cells has been obtained using fluorescence fluctuation spectroscopy (FFS). FFS provides a sensitive, specific and non-invasive tool to probe dynamics of fluorescent species with high spatial and temporal resolution in living cells.<o:p></o:p></span>Chapter 1:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>This chapter gives a brief introduction to the chemotaxis pathway of Dictyostelium and to the principle of FFS.<o:p></o:p></span>Chapter 2:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>Here the spectral properties of Dictyostelium autofluorescence are characterized to estimate the interference of autofluorescence in FFS measurements. It is seen that Dictyostelium autofluorescence is localized in discrete areas in both chemotaxing and non-chemotaxing cells and covers a wavelength range from ~ 500 - 650 nm with a maximum at ~ 510 nm. The cells exhibit continuous movement giving rise to slow non-uniform intensity fluctuations around the average intensity value in FFS measurements. The effect of the cell movement can be minimized by acquiring data for short times of 5 -10 s. This also helps in minimizing cellular photodamage during measurements. The most optimal conditions for performing FFS measurements in Dictyostelium cells are low excitation intensities (&lt;20</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>m</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>W), short measurement times and the use of prebleaching to reduce autofluorescence.<o:p></o:p></span>Chapter 3:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>The translocation of PH domain-containing proteins from the cytoplasm to the plasma membrane is thought to play an important role in the chemotaxis mechanism of Dictyostelium cells. This chapter describes the diffusion of three GFP tagged pleckstrin homology domains, PH-CRAC, PH2 and PH10 in the cell cytoplasm and how it is affected upon initiation of chemotaxis. A comparison has been made with the diffusion of free GFP in cells. It is observed that the various PH domains are differently affected by initiation of chemotaxis.<o:p></o:p></span>Chapter 4:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>The heterotrimeric G-proteins, composed of G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>a</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>, G</span><span lang=EN-US style='font-family: Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-ansi-language:EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and<span style='mso-bidi-font-weight: bold'>G</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol;mso-bidi-font-weight: bold'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>are crucial for the chemotactic signaling. Receptor-mediated activation of the G-protein, at the inner face of the plasma membrane, leads to the dissociation of the G</span><span lang=EN-US style='font-family:Symbol;mso-ansi-language:EN-US'>a</span><span lang=EN-US style='mso-ansi-language:EN-US'>subunit from the G</span><span lang=EN-US style='font-family:Symbol;mso-ansi-language:EN-US'>bg</span><span lang=EN-US style='mso-ansi-language:EN-US'>subunits, which can then modulate downstream effectors. In this chapter the diffusion and interaction of GFP tagged G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and<span class=GramE>G<span style='font-family: Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-char-type:symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type: symbol;mso-symbol-font-family:Symbol'>g</span></span>subunits in cytoplasm of chemotaxing and non-chemotaxing cells has</span>been studied in detail. Results indicate that G</span><span lang=EN-US style='font-family:Symbol; mso-ascii-font-family:"Times New Roman";mso-hansi-font-family:"Times New Roman"; mso-ansi-language:EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and<span style='mso-bidi-font-weight: bold'>G</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol;mso-bidi-font-weight: bold'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>are associated with each other in both chemotaxing and non-chemotaxing cells. The diffusion of the G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family: "Times New Roman";mso-ansi-language:EN-US;mso-char-type:symbol;mso-symbol-font-family: Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>complex in different regions of the cell cytoplasm is influenced by the initiation of chemotaxis. A mechanism relating these differences in diffusion to amplification of the chemotactic response is proposed.<o:p></o:p></span>Chapter 5:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>In this chapter the effect of deletion of Phlp1, a protein that is thought to modulate G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family: "Times New Roman";mso-ansi-language:EN-US;mso-char-type:symbol;mso-symbol-font-family: Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>activity in Dictyostelium , on G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and G</span><span lang=EN-US style='font-family: Symbol;mso-ansi-language:EN-US'>g</span><span lang=EN-US style='mso-ansi-language: EN-US'>dimerization is investigated. The results indicate that in the absence of Phlp1, G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and<span style='mso-bidi-font-weight: bold'>G</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family: "Times New Roman";mso-hansi-font-family:"Times New Roman";mso-ansi-language: EN-US;mso-char-type:symbol;mso-symbol-font-family:Symbol;mso-bidi-font-weight: bold'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>do not form a complex. It is proposed that Phlp1 may serve as a co-chaperone assisting the assembly of G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>and<span style='mso-bidi-font-weight:bold'>G</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol;mso-bidi-font-weight:bold'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>into a functional G</span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman"; mso-hansi-font-family:"Times New Roman";mso-ansi-language:EN-US;mso-char-type: symbol;mso-symbol-font-family:Symbol'><span style='mso-char-type:symbol; mso-symbol-font-family:Symbol'>b</span></span><span lang=EN-US style='font-family:Symbol;mso-ascii-font-family:"Times New Roman";mso-hansi-font-family: "Times New Roman";mso-ansi-language:EN-US;mso-char-type:symbol;mso-symbol-font-family: Symbol'><span style='mso-char-type:symbol;mso-symbol-font-family:Symbol'>g</span></span><span lang=EN-US style='mso-ansi-language:EN-US'>complex.<o:p></o:p></span>Chapter 6:</span></strong><span lang=EN-US style='mso-ansi-language:EN-US'>This chapter summarizes the findings in this thesis and discusses the use of FFS as a tool to probe protein dynamics in Dictyostelium cells