"Altering tumor cell scaffolding: Looking for cytoskeleton- damaging agents" Consolato Maria Sergi, MSc, MD, PhD Department of Laboratory Medicine and Pathology University of Alberta 1 Technology and The Future of Medicine

Outlines Eukaryotic Cells contain many organelles and a complex cytoskeleton. Cytoskeleton as scaffolding of a cell. Actin cytoskeleton is organized into bundles and networks of filaments. Intermediate filaments are dynamic polymeric in the cell. Role of signal-transduction pathways in cell locomotion and the organization of the cytoskeleton. Intermediate filaments in disease. Possible anti-cancer therapy.

Cytoskeleton 3

Microtubules Located in the cytoplasm of all eukaryotic cells 25 nm in diameter ( the thickest fiber type) Built up of Tubulin (β-tubulin & α-tubulin) Movements tracks Separation of the chromosomes Compression resistance 4

Microtubules: Centrosomes & Centrrioles Where the microtubules are initiated 5

Microtubules: Cell and cell’s organelles movements Cilia & Flagella: protrude from cell surface cilia: abundant number with 0.25μm in length flagella: usually 1 – few per cell with 2-20 μm in length “9+2” patterns 6

Microtubules: Cell and cell’s organelles movements Beating patterns in cilia and flagella 7

Microtubules: Cell and cell’s organelles movements Motor proteins: when the motor protein attaches to organelle, it has the ability to move the organelle along microtubule. 8

Microfilaments (Actin filaments) Built from actin molecules Mechanical support 3D Structural networks inside plasma membrane Cell motility: contractility of muscle cells Actin & Myosin: the contractile engine 9

Myosin : Motor protein 10

Actin filaments Actin cytoskeleton is organized into bundles and networks of filaments Plasma membrane support 11

Actin filaments Bundles: actin filaments are closely packed in parallel arrays Networks: actin filaments are crisscross and loosely packed. 3D structure = gel-like cytosol The filaments of the bundles and networks are connected by actin-cross-linking proteins 12

Actin filaments: actin-cross-linking proteins 13 The formation of the bundles or networks are highly depended on the flexibility of the cross-linking protein Actin bundles: e.g. fimbrin & α-actinin Actin networks: e.g. filamin & spectrin

Intermediate filaments (Ifs) Diameter: microtubules>intermediat filament> microfilaments More permanent fixtures of cells Keratins Don not have a role in cell motility 14

Intermediate filaments are dynamic polymeric in the cell 15

3D reconstruction of a cytoskeleton It is highly useful tool to fully understand the structural and mechanical properties of the cytoskeleton 16 Extraction of the graph structure from a detailed section of a secondary electron tomogram. (a) Detail of a tomogram after thresholding. (b) Skeleton of the foreground phase. (c) Extracted network graph after all processing steps, cross-links highlighted by spheres. Journal of Microscopy, Vol. 239, Pt , pp. 1–16

IF network in a detergent-extracted Panc 1 cell visualized at a magnification of 50,000. Filaments in some depth are clearly visible at good contrast in the secondary electron tomogram. The graph extracted from the tomogram exhibits a genuinely 3D structure. (a) SEM image at 0◦ tilt. (b) Horizontal cut through an SEM tomogram. (c) Vertical cut through an SEM tomogram. (d) Tomogram after binarisation (top view). (e) Network graph extracted from the tomogram, top view (left), rotated by 40◦ around central axis (right). 17 Journal of Microscopy, Vol. 239, Pt , pp. 1–16

3D reconstruction of a cytoskeleton 18 Reconstructed 3D cytoskeleton structure visualized by isosurface method. electron microscope stereo images (400]400 in pixels) of cell cytoskeleton taken at$103 of tilt angles Journal of Biomechanics 33 (2000) 105}113

Role of signal-transduction pathways in cell locomotion and the organization of the cytoskeleton 19

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Cont’d 21

Hedgehog (Hh) signaling in cancer Examples: Basal cell carcinomas Medulloblastomas 22

Inhibition of Hh-Gli-1signaling in cancers Inhibition of human prostate cancer cell proliferation by cyclopamine and GLI1 RNA interference. Cyclopamine inhibits the proliferation of primary in situ prostate tumors (A) and of metastatic prostate cancer cell lines (C), as measured by BrdU incorporation, and inhibits expression of GLI1 (B). Similarly, GLI1 small interfering RNAs (siRNAs) decrease GLI1 and PTCH1 expression (D,F) and inhibit the proliferation of prostate cancer cells (E). The cell line DU145 is not sensitive to cyclopamine while it is sensitive to GLI1 RNAi, suggesting activation of the pathway downstream of SMOH. The efficiency of GLI1 siRNAs in PC3 cells is low due to the low siRNA transfection efficiency. 23 Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience;

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