Fluorescence Microscopy to Investigate Cell-Cell …inbios21/PDF/Fall2008/Falk_12032008.pdf ·...

BioS95: Bioscience in the 21st Century, LU 12-03-08

Fluorescence Microscopy to InvestigateCell-Cell Communication

Matthias M. Falk, Ph.D.

Lehigh UniversityDepartment of Biological Sciences

Iacocca Hall, #D218111 Research Drive

Bethlehem, PA 18015610-758-5896

[email protected]://www3.lehigh.edu/~inbios/faculty/falk.htm

Figure 1-9Figure 1-6 ©M. Falk, All Rights Reserved

How big is a cell and how big are its components?

Light Microscope

X-ray CrystallographyElectron Microscope

Unaided Eye

©M. Falk, All Rights Reserved

Antonie van Leeuwenhoek, 1632-1723

Early Light Microscopes

Zeiss, ca, 1900

Compound

The resolution limit for self-luminousobjects is given in Rayleigh’s law:

d = 1.22 x λ

2 x NA

d = smallest resolvable distance between 2 points

λ = wavelength in nm (eGFPem. max. = 509 nm)

NA = Numerical Aperture (How much light a lens collects, high NA = high resolution)

d = 1.22 x 509

2 x 1.4

= 222 nm ©M. Falk, All Rights Reserved

BioS 368 Cell Biology Lab (Fall ‘05)



BioS 368 Cell Biology Lab (Fall ‘05)

Fluorescence Light Microscopes

Dr. Heinz Falk 1923 - 2001Siemens Electron Microscope



Cell CultureIncubator

(1) Cell Culture Techniques:Lots of cells in the incubator

©Image: LU BioS368, Instructor: Prof. M. Falk; all rights reserved

Human Epithelial Cells (HeLa), Bright Field


50 µm

HeLa CellsPhase Contrast Differential Interference Contrast, DIC

©M. Falk, All Rights Reserved50 µm

100 Trillion = 1014 cells/human body; >200 different cell types

From: Alberts et al. Molecular Biology of the Cell, 4th ed., Garland Sciences, 2002

Histochemical Stains of Tissue Sections

Skeletal Muscle


Fluorescent Dyes and their Excitation and Emission Wavelengths

DAPI

Fluorescein

Rhodamine©M. Falk, All Rights Reserved


Inverted

Mitochondria of a living HeLa cell stained with Mito Tracker Green.Note the long, tubular structure of the mitochondria in this cell type. The nucleus was stained withHoechst33342.


Endoplasmic reticulum (ER) stained with ER-Tracker Blue-White in a liveHeLa cell. Note the fine, tubular reticulum extending throughout the cytoplasm.


The Golgi apparatus of living HeLa cells stained with BODIPY TR-labeledceramide. Note the cap-like appearance of the Golgi cisternae on one side of the nucleus. Cellnuclei were stained with Hoechst33342.


Lysosomes stained in living HeLa cells with LysoTracker Red.Note the accumulation of lysosomes in peri-nuclear regions of the cytoplasm. Cell nuclei werestained with Hoechst33342.


DNA and RNA visualized in living HeLa cell. The dye acridine orange fluorescesred when bound to single stand nucleic acids, and green when bound to double-strand nucleicacids. Note the green nucleus (chromatin), the red hot-spots of mRNA translation in thecytoplasm, and the red-yellow nucleoli (ribosomal RNA synthesis centers) in the nucleus.


Stress-fibers assembled from bundles of actin filaments crossing thecell body were stained with Alexa488-labed phalloidin in fixed HeLacells. Phalloidin is a cyclic peptide that efficiently binds to actin filaments and interferes withtheir dynamics, making mushrooms of the Amanita family deadly poisonous. The nucleus was stainedwith DAPI.


Cell/extracellular matrix interactions (Focal Adhesions) visualized by stainingfixed HeLa cells with monoclonal anti-human vinculin (a protein of focal adhesion sites) and secondaryCy3-labeled goat anti-mouse specific antibodies, Alexa488-labeled phalloidin, and DAPI. Note thelocalization of vinculin at the end of stress-fibers especially in the cell periphery.


Cell-cell junctions (Adherens Junctions) visualized by staining fixed porcine PrimaryPulmonary Artery Endothelial cells (PAECs) with monoclonal anti-β-catenin (a focal adhesion protein)and secondary Alexa568-labeled goat anti-mouse specific antibodies, Alexa488-labeled phalloidin,and DAPI. Note the localization of β-catenin at the end of stress-fibers between cells.


Microtubules and centrosomes, the microtubule-organizing center(MTOC) in interphase HeLa cells. Microtubules were visualized using ananti-β-tubulin monoclonal antibody followed by Alexa488-labeled anti-mouse antibodies (green),and centrosomes were visualized using a γ-tubulin polyclonal antibody followed by Cy3-labeled antirabbit antibodies (red). Nuclei were labeled with DAPI.


Mitotic HeLa cells in Metaphase (left), early Anaphase (center), andCytokinesis (right). In Metaphase the condensed sister-chromosomes are aligned in thecenter of the spindle in the spindle plate, and in Anaphase are separated and moved to thespindle poles. Cytokinesis is the process of complete cell-separation.


Live-Cell Chamber on Microscope Stage


Hours:min

Human Cultured Cells (HeLa) Growing on a Glass Coverslip



Green Fluorescent Protein (GFP) and Derivatives

Roger Tsien, Nobel Prize in ChemistryWinner, 2008, UCSD

Expression of green (GFP), yellow (KO), and red fluorescent (DsRed)proteins in transiently transfected HeLa cells. Fluorescence image on the left, combined phase-contrast and fluorescence image on the right.


Ca2+Ca2+Electrical CurrentsElectrical Currents2nd Messengers (IP3)2nd Messengers (IP3)

ProteinsProteinsNucleic AcidsNucleic Acids

7nm 1.5nm

17 nm

3 nm

Gap Junction Structure and Function

Freeze Fracture

200 nm

Negative Stain

100 nm

EM Thin Section

(From: Falk, J. Cell Sci., 2000 Falk, Trends Cell Biol., 2002 Segretain & Falk, BBA, 2004)

100 nm

Cx43-GFP

Cx32-GFP

Gap Junction Plaques Assembled fromDifferent GFP-tagged Connexins

20

(From Falk, J. Cell Sci. 113:4109-20, 2000)

(Transfected HeLa cells)

Cx26-GFP

Volume Reconstruction of a Gap Junction Plaque

Unprocessed Deconvolved 3D-Volume Reconstruction

50 consecutive sections, z-steps 0.2 µm

(From Falk, J. Cell Sci.113:4109-20, 2000)

Objective lens

(not drawn to scale)

Focal plane

Z

Structural Composition of Gap Junction Plaques

(From Falk, J. Cell Sci. 113:4109-20, 2000)

SeparatedHomoHomomeric

Heteromeric

Channel Type and Arrangement:

91 frames, 10 sec apart, 15 min total time, looped

5 µm

Cx43-GFP

Structural Dynamics of Gap Junctions(Lateral Motion of GJ Channels in the Plane of the

Membrane; Surface View)

200(From Lopez et al., Cell Com. & Adhes., 8:237, 2001)

Edge-View:

Surface-View:

Movement of Cx-Containing Transport Vesicles near Gap Junction Plaques

250 frames, 2 sec apart, 8 min : 20 sec total time, looped

5

Cx43-GFP


5 µm

Trafficking of Exocytic and Endocytic Vesicular Connexin-Cargo along Microtubules


10

Cx43-CFPCx43-CFP / / LysoTrackerLysoTracker / / ββ--TubulinTubulin-YFP-YFP

(From Lauf, …, & Falk, PNAS 99,10446-10451, 2002)

10 µm


Cx43-CFP / β-Tubulin-YFP TP 1 - 21 (Merged)

TP 2 - 17

5 µm

Transport of Secretory Cx-Cargo along Microtubules


Gap Junction Channels are Recruited to the Edge of Channel Clusters(Fluorescence Recovery After Photobleaching, FRAP)

Cx43-GFP

(From Lauf et al., PNAS, 99:10446, 2002)

Surface Views

Edge Views


Gap Junction Channels are Recruited to the Edge of Channel Clusters(Photoconversion of Cx43-Dendra2)

1 hour post-conversion

5 µm

(From: Piehl et al.,MBC, 2007)

Entire Gap JunctionPlaques can Internalize to

form Annular GapJunctions (AGJs)

(Fluorecence Time LapseMicroscopy)

(Transfected HeLa cells)Hours:min

(Fluorescence and DIC)

Biosynthesis and Degradation of Gap Junctions(A Summary)

(From: Segretain & Falk, BBA, 2004)


Detection of Cells Growing on Non-Transparent Objects:MC3T3 bone-precursor cells growing on non-transparent bio-glass, (nuclei stained with DAPI, combined fluorescence and reflection illumination)


X-Z

Y-Z

1 of 50 sections

Projections: 3D Volume Reconstructions:Scanning EM:

Detection of Cells Growing on Non-Transparent Objects:MC3T3 bone-precursor cells growing on non-transparent bio-glass, (nuclei stained with Propidium Iodide, Actin decorated with Alexa488-Phalloidin

Thank You!Thank You!

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