In Vitro Methods for Investigating Desmoplakin–Intermediate Filament Interactions and Their Role in Adhesive Strength

Hudson, Tracie Y.; Fontao, Lionel; Godsel, Lisa M.; Huen, Auris; Borradori, Luca; Weis, William I.; Green, Kathleen J.

doi:10.1016/s0091-679x(04)78026-7

Cited by 24 publications

(28 citation statements)

References 29 publications

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“…(encompassing residues 2442-2630 of the linker region) did weakly bind to desmin only when tested as GAL4-AD fusion proteins against desmin fused to the GAL4-BD, as inferred from the activation of the reporter gene ADE2 only (Hudson et al, 2004). By contrast, DP-C S2849G , DP-C⌬51 as well as DP-Ct S2849G (consisting of the 79-residue-long C-terminal extremity of DP) never showed binding activity to desmin.…”

Section: S2849gmentioning

confidence: 96%

New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

Lapouge

Fontao

Champliaud

et al. 2006

Journal of Cell Science

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Desmosomes are intercellular adhesive complexes that anchor the intermediate filament cytoskeleton to the cell membrane in epithelia and cardiac muscle cells. The desmosomal component desmoplakin plays a key role in tethering various intermediate filament networks through its C-terminal plakin repeat domain. To gain better insight into the cytoskeletal organization of cardiomyocytes, we investigated the association of desmoplakin with desmin by cell transfection, yeast two-hybrid, and/or in vitro binding assays. The results indicate that the association of desmoplakin with desmin depends on sequences within the linker region and C-terminal extremity of desmoplakin, where the B and C subdomains contribute to efficient binding; a potentially phosphorylatable serine residue in the C-terminal extremity of desmoplakin affects its association with desmin; the interaction of desmoplakin with non-filamentous desmin requires sequences contained within the desmin C-terminal rod portion and tail domain in yeast, whereas in in vitro binding studies the desmin tail is dispensable for association; and mutations in either the C-terminus of desmoplakin or the desmin tail linked to inherited cardiomyopathy seem to impair desmoplakindesmin interaction. These studies increase our understanding of desmoplakin-intermediate filament interactions, which are important for maintenance of cytoarchitecture in cardiomyocytes, and give new insights into the molecular basis of desmoplakin- and desmin-related human diseases.

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Section: S2849gmentioning

confidence: 96%

New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

Lapouge

Fontao

Champliaud

et al. 2006

Journal of Cell Science

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“…In contrast, there was little impact on the extent to which cell-cell interfaces were occupied by E-cadherin, although both inhibitors resulted in a more organized appearance of this classic cadherin (Figure 1, A and B). To test whether Dsg2 redistribution correlated with an increase in intercellular adhesive strength, epithelial sheets treated with DMSO, PKI, or the broad spectrum MMP inhibitors GM6001 ( Figure 1C) or TAPI (not shown) were released with dispase and were subjected to mechanical shear stress as described (Hudson et al, 2004). Like EGFR inhibition, MMP inhibition increased epithelial sheet integrity in a calcium-dependent manner, even at the highest concentration, supporting the physiological relevance of MMP-dependent effects on intercellular adhesion ( Figure 1C).…”

Section: J L Klessner Et Almentioning

confidence: 99%

“…Released monolayers were either analyzed directly without applying mechanical stress or transferred to a 15-ml conical tube and washed with PBS (two times). Tubes were inverted (0.09 mM ϭ 0 inversions, 0.50 mM ϭ 5-10 inversions, 1.0 mM ϭ Ͼ10 inversions), and fragments were counted with a dissecting scope (Leica, MZ6) as described (Hudson et al, 2004) or imaged with a Hamamatsu Orca digital camera (model C4742-95) and analyzed using MetaVue imaging software (Universal Imaging). Data are expressed as average number of fragments Ϯ SD.…”

mentioning

confidence: 99%

EGFR and ADAMs Cooperate to Regulate Shedding and Endocytic Trafficking of the Desmosomal Cadherin Desmoglein 2

Klessner

Desai

Amargo³

et al. 2009

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Regulation of classic cadherins plays a critical role in tissue remodeling during development and cancer; however, less attention has been paid to the importance of desmosomal cadherins. We previously showed that EGFR inhibition results in accumulation of the desmosomal cadherin, desmoglein 2 (Dsg2), at cell-cell interfaces accompanied by inhibition of matrix metalloprotease (MMP)-dependent shedding of the Dsg2 ectodomain and tyrosine phosphorylation of its cytoplasmic domain. Here, we show that EGFR inhibition stabilizes Dsg2 at intercellular junctions by interfering with its accumulation in an internalized cytoplasmic pool. Furthermore, MMP inhibition and ADAM17 RNAi, blocked shedding and depleted internalized Dsg2, but less so E-cadherin, in highly invasive SCC68 cells. ADAM9 and 15 silencing also impaired Dsg2 processing, supporting the idea that this desmosomal cadherin can be regulated by multiple ADAM family members. In contrast, ADAM10 siRNA enhanced accumulation of a 100-kDa Dsg2 cleavage product and internalized pool of Dsg2. Although both MMP and EGFR inhibition increased intercellular adhesive strength in control cells, the response to MMP-inhibition was Dsg2-dependent. These data support a role for endocytic trafficking in regulating desmosomal cadherin turnover and function and raise the possibility that internalization and regulation of desmosomal and classic cadherin function can be uncoupled mechanistically. INTRODUCTIONThe ability of cells to modulate their contacts with each other and the underlying matrix is essential for epithelial remodeling that occurs in development and cancer progression (Behrens, 1999;Thiery, 2003;Kramer et al., 2005). In particular, members of cadherin family of calcium-dependent intercellular adhesion molecules have been demonstrated to both suppress (Frixen et al., 1991) and promote (Islam et al., 1996) cell migration and invasion. Although classic cadherins assemble into intercellular adhesive structures known as adherens junctions that associate with the cortical actin cytoskeleton, desmosomal cadherins, including desmogleins and desmocollins, make up the adhesive core of desmosomes, which anchor intermediate filaments (IF) to sites of strong intercellular adhesion (Green and Simpson, 2007).Anchorage to the IF cytoskeleton is established with the cooperation of the desmosomal cadherin-associated proteins plakoglobin and plakophilins, which in turn link the IF-associated protein desmoplakin (DP) to the membrane complex. Desmosomes provide mechanical integrity to epithelial and heart tissues by redistributing the forces of mechanical stress . In addition, desmosomal cadherins have more recently emerged as playing a role in tissue morphogenesis (Runswick et al., 2001;Chidgey and Dawson, 2007;Dusek et al., 2007).In spite of desmosomes' importance in tissue function, most studies have focused on the contribution of classic cadherins to epithelial remodeling. Classic cadherins engage in bidirectional signaling with receptor tyrosine kinases (RTKs), whereby they are both ...

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“…To elucidate whether the increase in p0071 and the modulation of AJs correlated with increased intercellular adhesive strength, we released epithelial sheets of control or Rab11a siRNAtransfected MCF-7 cells using dispase and subjected them to mechanical shear stress as previously described (Hudson et al, 2004). Whereas epithelial sheets of control cells lost their integrity after 30 minutes of shaking, epithelial sheets of Rab11a-depleted cells stayed intact (Fig.…”

Section: P0071 Regulates the Localization Of Rab11mentioning

confidence: 94%

“…Dispase assays were performed as described previously (Hudson et al, 2004). Epithelial sheets of siRNA-transfected HeLa cells were detached using 4 U/ml dispase in normal medium for 30 minutes at 37˚C.…”

Section: Dispase Assaymentioning

confidence: 99%

The armadillo protein p0071 is involved in Rab11-dependent recycling

Keil

Hatzfeld

2013

Journal of Cell Science

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ABSTRACTp0071 is an intercellular junction protein of the p120 catenin family. We have identified Rab11a as a novel interaction partner of p0071. p0071 interacted preferentially with active Rab11a. Knockdown experiments revealed an interdependent regulation of both proteins. On the one hand, p0071 depletion induced a perinuclear accumulation of Rab11, suggesting a role of p0071 in the anterograde transport of Rab11 from the pericentrosomal region to the plasma membrane but not in retrograde transport. p0071 as well as Rab11 depletion increased transferrin receptor recycling indicating that p0071-induced Rab11 mislocalization interfered with Rab11 function and shifted recycling from the slow Rab11-dependent pathway to the fast Rab4-dependent pathway. When p0071 or Rab11 depletion was combined with a Rab4 knockdown the effect was reversed. On the other hand, Rab11a depletion increased p0071 recycling to cell contacts thereby identifying p0071 as a Rab11 cargo protein. This correlated with increased intercellular adhesion. Thus, we propose that p0071 has a key role in regulating recycling through the Rab11-dependent perinuclear recycling compartment, and links the regulation of adherens junctions to recycling to allow dynamic modulation of intercellular adhesion.

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In Vitro Methods for Investigating Desmoplakin–Intermediate Filament Interactions and Their Role in Adhesive Strength

Cited by 24 publications

References 29 publications

New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

New insights into the molecular basis of desmoplakinand desmin-related cardiomyopathies

EGFR and ADAMs Cooperate to Regulate Shedding and Endocytic Trafficking of the Desmosomal Cadherin Desmoglein 2

The armadillo protein p0071 is involved in Rab11-dependent recycling

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