Transcript Looking for cytoskeleton-damaging agents

Technology and The Future of Medicine
"Altering tumor cell scaffolding:
Looking for cytoskeletondamaging agents"
Consolato Maria Sergi, MSc, MD, PhD
Department of Laboratory Medicine and Pathology
University of Alberta
1
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
Actin & Myosin: the contractile engine
 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
 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
13
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
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
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 1 2010, pp. 1–16
3D reconstruction of a cytoskeleton
electron microscope stereo images
(400]400 in pixels) of cell
cytoskeleton taken at$103 of tilt
angles
Reconstructed 3D cytoskeleton
structure visualized by isosurface
method.
18
Journal of Biomechanics 33 (2000) 105}113
Role of signal-transduction pathways in cell locomotion
and the organization of the cytoskeleton
19
20
Cont’d
21
Hedgehog (Hh) signaling in cancer
 Examples: Basal cell carcinomas
Medulloblastomas
22
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.
23
Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.
Thank you