Presentation : Isoltaion and characterization of cytoplasmic cofilin - Actin rods

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The Journal of Biological Chemistry
The Journal of Biological Chemistry
285, 5450-5460.
285, 5450-5460.
East Carolina University
IDPBS – Chemistry
Chem. 8810
Isolation and Characterization of
Cytoplasmic Cofilin-Actin Rods
Resources: THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 8, pp. 5450–5460, February 19, 2010
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Actin – Cofilin Rods
1- What are Actin – cofilin rods ?
2- The Research Paper
What is Actin ?
 Globular multi-functional protein
 Form microfilaments.
 Found in essentially all eukaryotic cells
 Could be :
1- Free monomer called G-actin (globular)
2- Linear polymer microfilament called F-actin (filamentous)
 Essential for
1- Mobility
2- Cell division.
3- Signaling
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What is Cofilin ?
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 Cofilin ( ADF ) is a family of actin-binding
proteins  disassembles actin filaments

depolymerization at the minus end of filaments
 preventing their reassembly
Resourse: https://www.mechanobio.info/cytoskeleton-dynamics/what-is-thecytoskeleton/what-are-actin-filaments/how-do-actin-filaments-depolymerize/
What are Cofilin – Actin Rods ?
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Form in axons and dendrites of stressed neurons
lead to synaptic dysfunction
mediate cognitive deficits in dementias
Back
The Research Paper
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Back
The Research Paper Outcomes
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1- Rods form in the cytoplasm in response to many treatments (ATP-depletion treatments) that induce rods in
neurons.
2- Isolated Rods are stable in dithiothreitol (DTT) , EGTA, Ca2+, and ATP. BUT the did not survive in Triton.
Cofilin-GFP-containing rods are stable in 500 mM NaCl  rods formed from endogenous proteins are
significantly less stable in high salt.
3- Rods contain ADF/cofilin and actin in a 1:1 ratio.
The Research Paper Outcomes
4- The average length distribution of filaments within a rod from an A4.8
5- Only actin and ADF/cofilin are in rods during all phases of their formation.
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Rods form in the cytoplasm in
response to many treatments that
induce rods in neurons.
1- Cell lines: A431 cells and HeLa
cells
ATP depletion induces rod formation in cultured cells.
a, XACGFP rods in A4.8 cells; b, cofilin-GFP rods in HeLa cells; c, cofilinimmunostained rods in A431 cells; d, cofilin-immunostained rods in primary cultures of
rat cortical neurons. All panels show inverted fluorescence images. Bars,10 µm.
2- Cells were washed with 3 × 10 ml
of calcium- and magnesium-free
phosphate-buffered saline (PBS) and
then ATP-depleted in 10mM sodium
azide, 6mM 2-deoxyglucose in PBS
for 1 h at 37 °C.
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Isolated Rods are stable
Exception: Triton
The
decreased
stability
of
rods
composed of endogenous proteins to
Stability of isolated rods to 10-min exposure to different reagents.
(A) Plots of relative rod index for ADF /cofilin-GFP-containing rods.
(B) Plots of relative rod index for cofilin-immunostained endogenous
Rods.
The cofilin-GFP rods in A are from HeLa cells, and the XAC-GFP rods are from A4.8 cells.
high salt suggests that the fluorescent
proteins play a role in stabilizing the
cofilin-GFP-actin rods.
stability results are the mean values of triplicate samples from a single experiment.
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1:1 – Actin Cofilin Ration
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Rods  ATP-depleted neurons + cofilin- GFPexpressing cells
Contain
ADF/ cofilin and actin in a ratio of 1:1;
Supporting this
Rod-like structures form in vitro when the two
proteins are incubated together in equal molar
ratios.
(a) Fluorescent rod-shaped structures formed
(b) That were also visible by phase contrast.
(e) Fluorescent filaments that looked like they
rod were observed.
were coalescing into a
Rods were not formed when actin was used in the
absence of cofilin.
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ATP depletion Time Course
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cofilin-actin assembles into filaments, which coalesce into bundles that
condense into rods.
A431 cells (top panels) were ATP-depleted and fixed at 5, 10, 20, and 30 min, followed by cofilin-immunostaining.
(Bottom panels) inverted time-lapse images of ATP-depleted HeLa cells expressing cofilin-GFP.
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Rods Average Length
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The average filament length in the rod is 250 nm, which corresponds to about 96 actin subunits in length
electron microscopic tomography was used
Tomographic analysis of rod in an A4.8 cell
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Rods Composition
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Proteomic analysis of rods showed the presence of many other proteins. However, none of the more
abundant contaminating proteins localized to rods during their initial formation, although some are
recruited after rod maturation.
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