title: Single particle localization and tracking resolves different Exo70 dynamics in non-polar cells and neurons
Creators: name: Gosain, Hiteshika
Publisher: name: University of Muenster, Institutional Repository MIAMI
Year: 2024
Subjects: subject: 570 Biology

description: Diese Dissertation untersucht die Dynamik von Exo70, einer entscheidenden Untereinheit des Exozyst-Komplexes, an der Plasmamembran mittels superauflösender Fluoreszenzbildgebung. Zunächst wird eine systematische Analyse der Lokalisierung und funktionellen Eigenschaften von Exo70 durchgeführt, wenn es entweder am N-Terminus oder am C-Terminus markiert ist. Durch Einzelpartikel-Verfolgung (SPT), Fluoreszenzlebensdauer-Bildgebung und Modellierung wird nachgewiesen, dass die Position des Tags die Funktion von Exo70 verändert. Anschließend wurde die regulatorische Rolle von TC10 auf die Dynamik von Exo70 untersucht, indem Exo70 gemeinsam mit TC10 und Mutanten von TC10 in nicht-polaren Zellen und Neuronen mittels SPT Quantifizierung exprimiert wurde. Diese Ergebnisse zeigten, dass die Funktionen von Exo70 zell- und kompartimentspezifisch sind. Schließlich werden die Interaktionen zwischen Exo70 und Sec3 mittels Zwei-Farben SPT und -Verfolgung sowie der Segmentierung der Spuren untersucht. Es wird gezeigt, dass nur Exo70 und nicht Sec3 als räumlicher Orientierungspunkt in HeLa-Zellen diente.
description: This dissertation investigates the dynamics of Exo70, a crucial subunit of the exocyst complex, at the plasma membrane using super-resolution fluorescence imaging. At first, a systematic analysis of the localization and functional properties of Exo70 when either tagged at its N-terminus or C-terminus is explored. Through single particle tracking (SPT), fluorescence lifetime imaging and modelling, it is demonstrated that the position of the tag alters the function of Exo70. Next, the regulatory role of TC10 on Exo70 dynamics was studied by co-expressing Exo70 with TC10 and mutants of TC10 with Exo70 in non-polar cells and neurons using SPT quantification. These results showed that Exo70 functions are cell and compartment specific. Finally, the interactions between Exo70 and Sec3, are explored using two-color SPT.
description: 1 The Exocyst Complex ..... 1
1.1. Introduction ..... 1
1.2. The Structure and Organization of the Exocyst Complex ..... 2
1.3. The Exocyst in Vesicle Tethering for Exocytosis ..... 3
1.4. Small GTPases Regulate Subunits of the Exocyst ..... 5
1.5. Exocyst Complex During Neuronal Development ..... 7
2 Objectives ..... 10
3 Super-Resolution Imaging in Living Cells ..... 12
3.1. Introduction ..... 12
3.2. Fluorescence Microscopy Techniques for Live Cell Imaging ..... 13
3.2.1. Confocal Microscopy ..... 13
3.2.2. Total Internal Reflection Microscopy ..... 14
3.2.3. Lattice Light-Sheet Microscopy ..... 15
3.2.4. Single Molecule Localization Microscopy ..... 17
3.2.5. Single Particle Tracking ..... 18
3.3. Analysis of Single Particle Localization and Tracking ..... 20
3.3.1. Characterizing the Readout of SPT ..... 21
3.3.2. Change of Diffusive State ..... 24
3.4. Fitting the MSD and Adding Experimental Limits ..... 26
3.5. Distribution of the Diffusion Coefficient ..... 27
3.6. Type of Diffusion at the Plasma Membrane ..... 28
4 Materials and Methods ..... 30
4.1. Materials Used ..... 30
4.2. Cell Culture and Cultivation ..... 36
4.3. Transfection ..... 37
4.4. Staining Cells for Live Cell Imaging ..... 38
4.4.1 HaloTag and SNAP-tag ..... 38
4.4.2 Labelling for Single Molecule Localization and Single Particle Tracking ..... 39
4.4.3 Labelling for Live Cell Imaging using Confocal Microscope ..... 40
4.5. Immunofluorescence Imaging ..... 40
4.6. Confocal and Fluorescence Lifetime Imaging ..... 41
4.7. Lattice-Sheet Imaging ..... 43
4.8. Epifluorescent and TIRF Imaging ..... 43
4.9. Dual Colour Image Alignment ..... 45
4.10. Single Particle Tracking Data Processing ..... 46
4.11. Generation of Particle Trajectories Using TrackMate ..... 47
4.12. MSD Analysis of Trajectories Generated by TrackMate ..... 48
4.13. Subclassifying motion from trajectories: using TraJClassifier ..... 49
4.14. Visual Method for Evaluating Colocalization ..... 49
4.15. 3D Homology Modelling ..... 51
5 Results ..... 52
5.1. Part 1: Localization and Dynamics of C and N-tagged Exo70 ..... 52
5.1.1. Localization of Endogenous Exo70 in HeLa Cells and Neurons ..... 52
5.1.2. Change in Exo70 Localization After Transfection ..... 54
5.1.3. High-Resolution Images Using Lattice-Sheet Microscope ..... 56
5.1.4. Effect of Tags on the Localization and Dynamics of Exo70 ..... 58
5.1.5. Differences in Fluorescence Lifetime Imaging of Exo70 ..... 63
5.1.6. 3D Homology Modelling of Tagged Exo70 ..... 65
5.2. Part 2: The Influence of Rho GTPase TC10 on Exo70 Dynamics ..... 67
5.2.1. Endogenous Involvement of TC10 with Exo70 in HeLa cells ..... 68
5.2.2. Localization of Exo70-HaloTag with SNAP-TC10 ..... 70
5.2.3. Dynamics and Localization of Exo70 Under the Influence TC10’s Mutants ..... 72
5.2.4. HeLa Cells: Super-resolution Analysis of Exo70 in Presence of TC10 and
Mutants of TC10 ..... 77
5.2.5. Cortical Neurons: Super-resolution Analysis of Exo70 in Presence of
TC10 and Mutants of TC10 in ..... 85
5.3. Part 3: Exo70’s Association with Sec3 at the Plasma Membrane in HeLa
cells ..... 95
5.3.1. Exo70 and Sec3 Interact Endogenously in HeLa Cells ..... 96
5.3.2. Exo70-HaloTag Interacts with Exocyst Subunit Sec3 Indicating
Exocytosis ..... 97
5.3.3. Quantification of Exo70-HaloTag and Sec3-SNAPtag Diffusion ..... 99
5.3.4. Influence on Dynamics by Mutating the PI(4,5)P2 Binding Site of Exo70 ..... 103
5.3.5. Exo70-K632,K635A-HaloTag also Recruits Sec3-SNAPtag for Possible
Exocytosis ..... 107
6 Discussion ..... 114
6.1. Part 1: Localization and Dynamics of C and N-tagged Exo70 ..... 114
6.2. Part 2: The Influence of Rho GTPase TC10 on Exo70 Dynamics ..... 116
6.3. Part 3: Exo70’s Association with Sec3 at the Plasma Membrane in HeLa
cells ..... 119
6.4. Conclusion ..... 120
6.5. A step beyond limitations: learning more from SPT ..... 123
7 Outlook ..... 125
8 Acknowledgement ..... 127
9 References ..... 129
Supplementary Material ..... i
S.1. Plasmid Maps ..... i
S.2. Acronyms ..... ii
S.3. List of Figures ..... iv
S.4. List of Tables ..... x
S.5. Declarations ..... xii
S.6. Curriculum Vitae ..... xiii