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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 expression of Syk dramatically altered the cellular topography, reduced cell height, increased elasticity, increased viscosity, and allowed visualization of a more substantial microtubule network.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 microtubulesrequires the catalytic activity ofSyk. (A) MDA-MB-231 cells expressing Syk-EGFP (Syk) or Syk-EGFP(K396R)(KD-Syk) and treated with nocodazole were stained with an antibodyagainst α-tubulin and a fluorescently tagged secondary antibody,and with 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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fig4: Stabilization of microtubulesrequires the catalytic activity ofSyk. (A) MDA-MB-231 cells expressing Syk-EGFP (Syk) or Syk-EGFP(K396R)(KD-Syk) and treated with nocodazole were stained with an antibodyagainst α-tubulin and a fluorescently tagged secondary antibody,and with 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 determine if Syk-dependentprotein phosphorylation was involvedin modulating cytoskeletal dynamics, we asked if the catalytic activityof Syk was required for the promotion of microtubule stability. Wegenerated a line of MDA-MB-231 cells in which the expression of acatalytically inactive version of Syk-EGFP [Syk-EGFP(K396R)] was underthe control of a tetracycline-inducible promoter. Cells induced toexpress Syk-EGFP or Syk-EGFP(K396R) were treated with nocodazole (10μM) for 30 min, fixed, immunostained with an α-tubulinantibody, and examined under the fluorescence microscope. There wasan obvious difference in the morphology of drug-treated cells expressingactive versus inactive kinase (Figure 4). Ascompared to cells expressing Syk-EGFP, Syk-EGFP(K396R)-expressingcells were more rounded because of microtubule depolymerization andresembled cells that lacked Syk. Thus, the ability of Syk to stabilizemicrotubules was a function of its catalytic activity.


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 microtubulesrequires the catalytic activity ofSyk. (A) MDA-MB-231 cells expressing Syk-EGFP (Syk) or Syk-EGFP(K396R)(KD-Syk) and treated with nocodazole were stained with an antibodyagainst α-tubulin and a fluorescently tagged secondary antibody,and with 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

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

fig4: Stabilization of microtubulesrequires the catalytic activity ofSyk. (A) MDA-MB-231 cells expressing Syk-EGFP (Syk) or Syk-EGFP(K396R)(KD-Syk) and treated with nocodazole were stained with an antibodyagainst α-tubulin and a fluorescently tagged secondary antibody,and with 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 determine if Syk-dependentprotein phosphorylation was involvedin modulating cytoskeletal dynamics, we asked if the catalytic activityof Syk was required for the promotion of microtubule stability. Wegenerated a line of MDA-MB-231 cells in which the expression of acatalytically inactive version of Syk-EGFP [Syk-EGFP(K396R)] was underthe control of a tetracycline-inducible promoter. Cells induced toexpress Syk-EGFP or Syk-EGFP(K396R) were treated with nocodazole (10μM) for 30 min, fixed, immunostained with an α-tubulinantibody, and examined under the fluorescence microscope. There wasan obvious difference in the morphology of drug-treated cells expressingactive versus inactive kinase (Figure 4). Ascompared to cells expressing Syk-EGFP, Syk-EGFP(K396R)-expressingcells were more rounded because of microtubule depolymerization andresembled cells that lacked Syk. Thus, the ability of Syk to stabilizemicrotubules was a function of its catalytic activity.

Bottom Line: 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.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