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Nanomechanical property maps of breast cancer cells as determined by multiharmonic atomic force microscopy reveal Syk-dependent changes in microtubule stability mediated by MAP1B.

Krisenko MO, Cartagena A, Raman A, Geahlen RL - Biochemistry (2014)

Bottom Line: The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells.Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity.This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡School of Mechanical Engineering, §Purdue Center for Cancer Research, and ∥Birck Nanotechnology Center, Purdue University , West Lafayette, Indiana 47907, United States.

ABSTRACT
The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.

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Stabilization of microtubules through the expression of Syk. (A)MDA-MB-231 cells lacking Syk (−Syk) or expressing Syk (+Syk)and treated with nocodazole were stained with an antibody againstα-tubulin and a fluorescently tagged secondary antibody, andwith DAPI to mark the nucleus. Cells were examined by phase contrastand fluorescence microscopy to detect α-tubulin (red), nuclei(blue), and Syk-EGFP (green). (B) Distances from the cell nucleusto the cell boundary as marked by α-tubulin fluorescence. Cellswere grouped into three categories as indicated. The bar is 10 μm.
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fig2: Stabilization of microtubules through the expression of Syk. (A)MDA-MB-231 cells lacking Syk (−Syk) or expressing Syk (+Syk)and treated with nocodazole were stained with an antibody againstα-tubulin and a fluorescently tagged secondary antibody, andwith DAPI to mark the nucleus. Cells were examined by phase contrastand fluorescence microscopy to detect α-tubulin (red), nuclei(blue), and Syk-EGFP (green). (B) Distances from the cell nucleusto the cell boundary as marked by α-tubulin fluorescence. Cellswere grouped into three categories as indicated. The bar is 10 μm.

Mentions: To study in moredetail possible effects of Syk on microtubules, we examined the stabilityof the microtubule network. MDA-MB-231 cells either lacking or expressingSyk-EGFP were treated with low concentrations of nocodazole for 30min to promote microtubule depolymerization. It has been shown thatdifferences in the stability of the microtubule network manifest themselvesas changes in resistance to nocodazole-induced depolymerization.39 Cells were fixed and stained with an antibodyagainst α-tubulin and examined by fluorescence microscopy. Therewas a pronounced difference in the morphology of the nocodazole-treatedcells between those lacking Syk, in which the microtubule networkwas largely disassembled, and those expressing Syk-EGFP, in whichcells retained microtubule structures resistant to drug-induced depolymerization(Figure 2A). To quantify these differences,we determined the persistence of the microtubule network by measuringthe distance of α-tubulin staining from the nucleus to the perimeterof each cell. Results from a comparison of more than 200 cells areshown in Figure 2B. Nocodazole-treated cellsexpressing Syk-EGFP exhibited a much greater level of retention ofα-tubulin-containing polymers than did cells lacking Syk. Incontrast to the expression of Syk, the overexpression of c-Src, whichtypically enhances cell motility,40 failedto prevent cells from rounding up following treatmentwith nocodazole (Figure S3 of the Supporting Information).


Nanomechanical property maps of breast cancer cells as determined by multiharmonic atomic force microscopy reveal Syk-dependent changes in microtubule stability mediated by MAP1B.

Krisenko MO, Cartagena A, Raman A, Geahlen RL - Biochemistry (2014)

Stabilization of microtubules through the expression of Syk. (A)MDA-MB-231 cells lacking Syk (−Syk) or expressing Syk (+Syk)and treated with nocodazole were stained with an antibody againstα-tubulin and a fluorescently tagged secondary antibody, andwith DAPI to mark the nucleus. Cells were examined by phase contrastand fluorescence microscopy to detect α-tubulin (red), nuclei(blue), and Syk-EGFP (green). (B) Distances from the cell nucleusto the cell boundary as marked by α-tubulin fluorescence. Cellswere grouped into three categories as indicated. The bar is 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4295795&req=5

fig2: Stabilization of microtubules through the expression of Syk. (A)MDA-MB-231 cells lacking Syk (−Syk) or expressing Syk (+Syk)and treated with nocodazole were stained with an antibody againstα-tubulin and a fluorescently tagged secondary antibody, andwith DAPI to mark the nucleus. Cells were examined by phase contrastand fluorescence microscopy to detect α-tubulin (red), nuclei(blue), and Syk-EGFP (green). (B) Distances from the cell nucleusto the cell boundary as marked by α-tubulin fluorescence. Cellswere grouped into three categories as indicated. The bar is 10 μm.
Mentions: To study in moredetail possible effects of Syk on microtubules, we examined the stabilityof the microtubule network. MDA-MB-231 cells either lacking or expressingSyk-EGFP were treated with low concentrations of nocodazole for 30min to promote microtubule depolymerization. It has been shown thatdifferences in the stability of the microtubule network manifest themselvesas changes in resistance to nocodazole-induced depolymerization.39 Cells were fixed and stained with an antibodyagainst α-tubulin and examined by fluorescence microscopy. Therewas a pronounced difference in the morphology of the nocodazole-treatedcells between those lacking Syk, in which the microtubule networkwas largely disassembled, and those expressing Syk-EGFP, in whichcells retained microtubule structures resistant to drug-induced depolymerization(Figure 2A). To quantify these differences,we determined the persistence of the microtubule network by measuringthe distance of α-tubulin staining from the nucleus to the perimeterof each cell. Results from a comparison of more than 200 cells areshown in Figure 2B. Nocodazole-treated cellsexpressing Syk-EGFP exhibited a much greater level of retention ofα-tubulin-containing polymers than did cells lacking Syk. Incontrast to the expression of Syk, the overexpression of c-Src, whichtypically enhances cell motility,40 failedto prevent cells from rounding up following treatmentwith nocodazole (Figure S3 of the Supporting Information).

Bottom Line: The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells.Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity.This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, ‡School of Mechanical Engineering, §Purdue Center for Cancer Research, and ∥Birck Nanotechnology Center, Purdue University , West Lafayette, Indiana 47907, United States.

ABSTRACT
The Syk protein-tyrosine kinase, a well-characterized modulator of immune recognition receptor signaling, also plays important, but poorly characterized, roles in tumor progression, acting as an inhibitor of cellular motility and metastasis in highly invasive cancer cells. Multiharmonic atomic force microscopy (AFM) was used to map nanomechanical properties of live MDA-MB-231 breast cancer cells either lacking or expressing Syk. The expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network. The microtubules of Syk-expressing cells were more stable to nocodazole-induced depolymerization and were more highly acetylated than those of Syk-deficient cells. Silencing of MAP1B, a major substrate for Syk in MDA-MB-231 cells, attenuated Syk-dependent microtubule stability and reversed much of the effect of Syk on cellular topography, stiffness, and viscosity. This study illustrates the use of multiharmonic AFM both to quantitatively map the local nanomechanical properties of living cells and to identify the underlying mechanisms by which these properties are modulated by signal transduction machinery.

Show MeSH
Related in: MedlinePlus