Resolving Macromolecular Organization in Cells Using Super Resolution Microscopy Karen Porter-Davis Chamblee Charter High School

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1 Resolving Macromolecular Organization in Cells Using Super Resolution Microscopy Karen Porter-Davis Chamblee Charter High School STEP-UP Program 2015

2 Super Resolution Imaging } Two Projects to Image } The relationship of actin and myosin II during frustrated phagocytosis. } The study of peri-cellular matrix (PCM) and cell adhesion, specifically the roles of hyaluronan (HA) and proteoglycan. PCM Fig. 1: Phagocytosis video from Fig. 2: Prostate cell; highlighting the PCM. Courtesy of Patrick Change

3 Immune Cell Fig. 3: Diagram of the IC binding with the Fc- Receptors on the Phagocyte. Image courtesy of the Curtis Group.

4 Actin } A protein found in many forms and has many functions within animal cells. } F-actin = Filament actin (forms microfilaments) } Actin that makes up the cytoskeleton: 1) stabilizes cell shape 2) assist in cell movement (whole cell or parts of the cell)

5 Myosin } Are a group of ATP-dependent motor proteins. } They are well known for their role in eukaryotic motility processes and muscle contraction. } Responsible for actin-based motility. } The structure and function of myosin is strongly conserved across species.

6 Pericellular Matrix (PCM): Polymer matrix grafted onto the cell surface Plasma membrane Hyaluronan (HA) 400 nm Proteoglycan Cell interior ) Fig. 4: PCM Boehm Soft Matter (2009); McLane, Chang, et al Biophysical J. (2013)

7 PCM is Important for Adhesion } Adhesion dependent processes } Proliferation } Migration } Synaptogenesis Fig. 5: Courtesy of Louis McLane Focal adhesion

8 Why Fluorescent Markers and Super-Resolution Microscopy? } Fluorescent microscopy has revolutionized biological sciences by allowing researchers to view cells and cellular components with a great amount of distinction compared to the non-fluorescing material. Fig. 6: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC), cell membrane with WGA633 and the nucleus with DAPI. Imaged with an Olympus Confocal Microscope Georgia Institute of Technology 2014 Fig. 7: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC) and the nucleus with DAPI. Preparallel bundling of the actin fibers around perimeter. Images created by K. Porter-Davis at Georgia Tech Microscopy Lab Summer 2014.

9 } Emission of light by a material that has absorbed light. The emitted light has a longer wavelength (lower energy) than the absorbed (UV à visible) } Ex)some minerals and gems } Re-emits in less than 10 ns Fig. 8: The photograph shows a collection of fluorescent minerals. It was created by Dr. Hannes Grobe and is part of the Wikimedia Commons collection. The photo is used here under a Creative Commons license. Fluorescence

10 The Jablonski Diagram Fig. 9: Jablonski Diagram. These notes were written by Dr. Thomas G. Chasteen at Sam Houston State University, Huntsville, Texas.

11 Frustrated Phagocytosis Image Fig. 10: Macrophage undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC) and the nucleus with DAPI. Twenty minute time; the cell is retracting. Image created by K. Porter-Davis at Georgia Tech Microscopy Lab Summer 2015.

12 PCM Image Fig. 13: Human prostate cell. An anti-vinculin protein is used to fluorescently label the cell to the focal adhesions and Green Fluorescent Protein (GFP) dyes the hyaluronan polymer chain of the matrix. Image created by K. PorterDavis at Georgia Tech Microscopy Lab Summer 2015.

13 Results and Future Work } The actin banding seen in our images lends support to the proposed mechanism of the constriction around the target particle. } Effectively label myosin II and the cell membrane to further understand the mechanics behind phagocytosis.

14 Old Protocol for Labeling Myosin Fig. 10: Macrophages undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC), the nucleus with DAPI and rabbit anti-myosin II A (H and L) antibody and donkey anti-rabbit IgG to label the myosin. Note issue with red labeling.

15 Processed Cell Fig. 11: Macrophages undergoing frustrated phagocytosis; labeled actin cytoskeleton with phalloidin (FITC), the nucleus with DAPI and rabbit anti-myosin II A (H and L) antibody and donkey anti-rabbit IgG to label the myosin. Much more specific labeling. 10 µm

16 Conclusion and Future Work } PCM acts as a mechanical repulsive cushion for adhesion } Cell adhesion strength versus PCM states } Spatial distribution of PCM patches and focal contacts } PCM mediated adhesion: mechanical or via signaling } PCM role in adhesion of cancer metastasis Fig. 12: Karen Porter-Davis

17 Lesson Plan } Fluorescence PowerPoint } Growing Fluorescent Crystals } Extraction of Chlorophyll } Glowing Flowers Fig. 13: p/black-light-projects.htm Fig. 14: Fig. 15: wing-flower.htm

18 Fig. 15: A glowing (GFP) kitten stands next to a normal cat. Photo courtesy of the Mayo Clinic.

19 ACKNOWLEDGEMENTS Thank you for the guidance and support: Dr. Leyla Conrad and the STEP-UP Program participants Dr. Jennifer Curtis Patrick Chang Jan Toro Wenbin Wei Andrew Shaw