"altering tumor cell scaffolding: looking for cytoskeleton- damaging agents" consolato...
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"Altering tumor cell scaffolding: Looking for cytoskeleton-
damaging agents"
Consolato Maria Sergi, MSc, MD, PhDDepartment of Laboratory Medicine and Pathology
University of Alberta
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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
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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
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Microtubules: Centrosomes & Centrrioles
Where the microtubules are initiated
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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 μmin length • “9+2” patterns
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Microtubules: Cell and cell’s organelles movements
Beating patterns in cilia and flagella
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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.
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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
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Actin & Myosin: the contractile engine
Myosin : Motor protein
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Actin filaments
Actin cytoskeleton is organized into bundles and networks of filaments
Plasma membrane support
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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
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Actin filaments: actin-cross-linking proteins
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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
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Intermediate filaments are dynamic polymeric in the cell
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3D reconstruction of a cytoskeleton
It is highly useful tool to fully understand the structural and mechanical properties of the cytoskeleton
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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 1 2010, 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).
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Journal of Microscopy, Vol. 239, Pt 1 2010, pp. 1–16
3D reconstruction of a cytoskeleton
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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
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Cont’d
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Hedgehog (Hh) signaling in cancer
Examples: Basal cell carcinomas Medulloblastomas
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http://www.med.umich.edu/derm/research/res_embryonic.shtml
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.
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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.
Thank you
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