Practical applications of TIRF microscopy Evgeny Pryazhnikov University of Helsinki, Neuroscience Center
Functional and Morphological Plasticity of the Tripartite Synapse Vesicular release ATP Perisynaptic astrocyte ATP, ROS Presynaptic neuron KAR Ca 2+ Glu, Ca 2+ Ca 2+ Mitochondrial motility Ca 2+ (LTP/LTD) Cl - (inhib./excit.) KCC2 Postsynaptic neuron Perisynaptic astrocyte Morph. plasticity Membrane trafficking Presynaptic neuron: ROS on GABA release ATP/adenosine on glutamate release KAR modulation of glutamate release Postsynaptic neuron: KCC2 regulation and membrane trafficking Cl - -dependent inhibition/excitation KCC2 in dendritic spines ATP on P2 receptors Perisynaptic astrocyte: Vesicular release of ATP Morphological plasticity of fine processes Mitochondrial motility and Ca 2+ signaling ROS generation and release
Part I Vesicles: following exocytosis
Vesicles in cultured astrocytes Pryazhnikov and Khiroug, Glia, 2008
Docking event Combination I: TIRF + EPI Criteria of docking and fusion TIRF field Fusion event TIRF field Pryazhnikov and Khiroug, Glia, 2008
Examples of spontaneous docking and undocking undocking event docking event TIRF + EPI Pryazhnikov and Khiroug, unpublished
Tracking the directional vesicles Pryazhnikov and Khiroug, Glia, 2008
Vesicular ATP release from astrocytes control upon stimulation TIRF EPI TIRF EPI Pryazhnikov and Khiroug, Glia 2008
Combination II: TIRF + caged Ca 2+ photolysis EGTA o-nitrophenyl EGTA (NP-EGTA) Upon UV illumination, the Ca 2+ -NPEGTA complex is cleaved to yield free Ca 2+ and two iminodiacetic acid photoproducts. The affinity of the photoproducts for Ca 2+ is ~12,500-fold lower than that of NP-EGTA http://www.invitrogen.com/
Combination II: TIRF + caged Ca 2+ photolysis measure Ca 2+ with Fura-2 dye EPI imaging follow exocytosis with quinacrine TIRF imaging Pryazhnikov and Khiroug, Glia, 2008
Vesicle behavior near plasma membrane Jaiswal and Simon, Nature Chem Biology, 2007
Different modes of exocytosis Pryazhnikov and Khiroug, Glia, 2008
Combination III: TIRF and phluorin tagging. TIRF and insulin exocytosis. Different modes of kiss-and-run Tsuboi and Rutter, Biochem Soc Trans, 2003
Part II Translocation of organelles towards the plasma membrane
Why and how do mitochondria move? MitoTracker Green FM1-43 (membrane)
Mitochondrion Plasma membrane Laser beam 100 nm Glass cover slip Evanescent Field, d p ~150 nm
Can TIRF see mitochondria? Epi TIRF Evanescent field Glass cover slip In Epifluorescence, all mitochondria are visible irregardless of their vertical position. Under TIRF illumination, only those mitochondria that are within ~100 nm from plasma membrane are visible. Kolikova et al. (2006) BCB
TIRF reveals mitochondrial vertical motility Epi TIRF Evanescent field Glass cover slip In Epifluorescence, all mitochondria are visible irregardless of their vertical position. Under TIRF illumination, only those mitochondria that are within ~100 nm from plasma membrane are visible. Kolikova et al. (2006) BCB
Mitochondria move to plasma membrane TIRF Control solution Epi TIRF +Epi Kolikova et al. (2006) BCB
Mitochondria move to plasma membrane Glutamate (0.1 mm, 1 min) TIRF Epi TIRF +Epi Kolikova et al. (2006) BCB
Distance between mito and PM shortens? Glu Mito TIRF Mito Epi 10 % PM TIRF 60 s Plasma membrane Mitochondria Kolikova et al. (2006) BCB
Ca 2+ influx induced trapping of mitochondria near plasma membrane Resting conditions Under stimulation TIRF + Epi Kolikova et al., BCB 2006
Mitochondrion Microtubule Plasma membrane Ca 2+
ER and plasma membrane Luik et al., Journal of Cell Biology, 2006
Secretory organelles and microtubules in endothelial cells Manneville et al., Journal of Cell Science, 2003
Combination IV: TIRF and FRAP TIRFRAP FRAP = Fluorescent Recovery After Photobleaching TIRF
Combination IV: TIRF and FRAP TIRFRAP SAP97-EGFP (TIRF+Epi) Kv4.2-EGFP (TIRF+Epi) Pryazhnikov et al., submitted
Only for cultured cells
Conclusions TIRF is suitable for studying: - stages of exo- and endocytosis of secreting organelles - trafficking of molecules and organelles to and from the cellular plasma membrane - binding of ligands to their membrane-bound receptors - dynamic lateral rearrangement of molecules within biomembranes
Acknowledgments Neuroscience Center Leonard Khiroug Sergey Khiroug Julia Kolikova Research was supported by CIMO, Academy of Finland and Finnish Cultural Foundation