The juxtamembrane domain of the E-cadherin cytoplasmic tail contributes to its interaction with Myosin VI.

We recently identified the atypical myosin, Myosin VI, as a component of epithelial cell-cell junctions that interacts with E-cadherin. Recombinant proteins bearing the cargo-binding domain of Myosin VI (Myo VI-CBD) or the cytoplasmic tail of E-cadherin can interact directly with one another. In this report we further investigate the molecular requirements of the interaction between Myo VI-CBD and E-cadherin combining truncation mutation analysis with in vitro binding assays. We report that a short (28 amino acid) juxtamembrane region of the cadherin cytoplasmic tail is sufficient to bind Myo VI-CBD. However, central regions of the cadherin tail adjacent to the juxtamembrane sequence also display binding activity for Myo VI-CBD. It is therefore possible that the cadherin tail bears two binding sites for Myosin VI, or an extended binding site that includes the juxtamembrane region. Nevertheless, our biochemical data highlight the capacity for the juxtamembrane region to interact with functionally-significant cytoplasmic proteins.

Multicomponent analysis of junctional movements regulated by myosin II isoforms at the epithelial zonula adherens.

The zonula adherens (ZA) of epithelial cells is a site of cell-cell adhesion where cellular forces are exerted and resisted. Increasing evidence indicates that E-cadherin adhesion molecules at the ZA serve to sense force applied on the junctions and coordinate cytoskeletal responses to those forces. Efforts to understand the role that cadherins play in mechanotransduction have been limited by the lack of assays to measure the impact of forces on the ZA. In this study we used 4D imaging of GFP-tagged E-cadherin to analyse the movement of the ZA. Junctions in confluent epithelial monolayers displayed prominent movements oriented orthogonal (perpendicular) to the ZA itself. Two components were identified in these movements: a relatively slow unidirectional (translational) component that could be readily fitted by least-squares regression analysis, upon which were superimposed more rapid oscillatory movements. Myosin IIB was a dominant factor responsible for driving the unilateral translational movements. In contrast, frequency spectrum analysis revealed that depletion of Myosin IIA increased the power of the oscillatory movements. This implies that Myosin IIA may serve to dampen oscillatory movements of the ZA. This extends our recent analysis of Myosin II at the ZA to demonstrate that Myosin IIA and Myosin IIB make distinct contributions to junctional movement at the ZA.

Hepatocyte growth factor acutely perturbs actin filament anchorage at the epithelial zonula adherens.

Cadherin adhesion molecules function in close cooperation with the actin cytoskeleton. At the zonula adherens (ZA) of polarized epithelial cells, E-cadherin adhesion induces the cortical recruitment of many key cytoskeletal regulators, which act in a dynamic integrated system to regulate junctional integrity and cell-cell interactions. This capacity for the cytoskeleton to support the ZA carries the implication that regulators of the junctional cytoskeleton might also be targeted to perturb junctional integrity. In this report, we now provide evidence for this hypothesis. We show that hepatocyte growth factor (HGF), which is well-known to disrupt cell-cell interactions, acutely perturbs ZA integrity much more rapidly than generally appreciated. This is accompanied by significant loss of junctional F-actin, a process that reflects loss of filament anchorage at the junctions. We demonstrate that this involves uncoupling of the unconventional motor myosin VI from junctional E-cadherin, a novel effect of HGF that is mediated by intracellular calcium. We conclude that regulators of the junctional cytoskeleton are likely to be major targets for cadherin junctions to be acutely modulated in development and perturbed in disease.

Motor training of upper extremity with functional electrical stimulation in early stroke rehabilitation.

BACKGROUND: Functional electrical stimulation (FES) allows active exercises in stroke patients with upper extremity paralysis. OBJECTIVE: To investigate the effect of motor training with FES on motor recovery in acute and subacute stroke patients with severe to complete arm and/or hand paralysis. METHODS: For this pilot study, 23 acute and subacute stroke patients were randomly assigned to the intervention (n = 12) and control group (n = 11). Distributed over 4 weeks, FES training replaced 12 conventional training sessions in the intervention group. An Extended Barthel Index (EBI) subscore assessed the performance of activities of daily living (ADL). The Chedoke McMaster Stroke Assessment (CMSA) measured hand and arm function and shoulder pain. The Modified Ashworth Scale (MAS) assessed resistance to passive movement. Unblinded assessments were performed prior to and following the end of the training period. RESULTS: The EBI subscore and CMSA arm score improved significantly in both groups. The CMSA hand function improved significantly in the FES group. Resistance to passive movement of finger and wrist flexors increased significantly in the FES group. Shoulder pain did not change significantly. None of the outcome measures, however, demonstrated significant gain differences between the groups. CONCLUSIONS: We did not find clear evidence for superiority or inferiority of FES. Our findings, and those of similar trials, suggest that the number of sessions should be at least doubled to test for superiority of FES in these highly impaired patients and approximately 50 participants would have to be assigned to each therapeutic intervention to find significant differences.