FE65 and FE65L1 share common synaptic functions and genetically interact with the APP family in neuromuscular junction formation.

The FE65 adaptor proteins (FE65, FE65L1 and FE65L2) bind proteins that function in diverse cellular pathways and are essential for specific biological processes. Mice lacking both FE65 and FE65L1 exhibit ectopic neuronal positioning in the cortex and muscle weakness. p97FE65-KO mice, expressing a shorter FE65 isoform able to bind amyloid precursor protein family members (APP, APLP1, APLP2), develop defective long-term potentiation (LTP) and aged mice display spatial learning and memory deficits that are absent from young mice. Here, we examined the central and peripheral nervous systems of FE65-KO, FE65L1-KO and FE65/FE65L1-DKO mice. We find spatial learning and memory deficits in FE65-KO and FE65L1-KO mice. Severe motor impairments, anxiety, hippocampal LTP deficits and neuromuscular junction (NMJ) abnormalities, characterized by decreased size and reduced apposition of pre- and postsynaptic sites, are observed in FE65/FE65L1-DKO mice. As their NMJ deficits resemble those of mutant APP/APLP2-DKO mice lacking the FE65/FE65L1 binding site, the NMJs of APLP2/FE65-DKO and APLP2/FE65L1-DKO mice were analyzed. NMJ deficits are aggravated in these mice when compared to single FE65- and FE65L1-KO mice. Together, our data demonstrate a role for FE65 proteins at central and peripheral synapses possibly occurring downstream of cell surface-associated APP/APLPs.

Adhesion maturation of neutrophils on nanoscopically presented platelet glycoprotein Ibα.

Neutrophilic granulocytes play a fundamental role in cardiovascular disease. They interact with platelet aggregates via the integrin Mac-1 and the platelet receptor glycoprotein Ibα (GPIbα). In vivo, GPIbα presentation is highly variable under different physiological and pathophysiological conditions. Here, we quantitatively determined the conditions for neutrophil adhesion in a biomimetic in vitro system, which allowed precise adjustment of the spacings between human GPIbα presented on the nanoscale from 60 to 200 nm. Unlike most conventional nanopatterning approaches, this method provided control over the local receptor density (spacing) rather than just the global receptor density. Under physiological flow conditions, neutrophils required a minimum spacing of GPIbα molecules to successfully adhere. In contrast, under low-flow conditions, neutrophils adhered on all tested spacings with subtle but nonlinear differences in cell response, including spreading area, spreading kinetics, adhesion maturation, and mobility. Surprisingly, Mac-1-dependent neutrophil adhesion was very robust to GPIbα density variations up to 1 order of magnitude. This complex response map indicates that neutrophil adhesion under flow and adhesion maturation are differentially regulated by GPIbα density. Our study reveals how Mac-1/GPIbα interactions govern cell adhesion and how neutrophils process the number of available surface receptors on the nanoscale. In the future, such in vitro studies can be useful to determine optimum therapeutic ranges for targeting this interaction.

Investigating cell-ECM contact changes in response to hypoosmotic stimulation of hepatocytes in vivo with DW-RICM.

Hepatocyte volume regulation has been shown to play an important role in cellular metabolism, proliferation, viability and especially in hepatic functions such as bile formation and proteolysis. Recent studies on liver explants led to the assumption that cell volume changes present a trigger for outside-in signaling via integrins, a protein family involved in mediating cellular response to binding to the extracellular matrix (ECM). However, it remains elusive how these volume change related signaling events are transducted on a single cell level and how these events are influenced and controlled by ECM interactions. One could speculate that an increase in cell volume leads to an increase in integrin/ECM contacts which causes activation of integrins, which act as mechano-sensors. In order to test this idea, it was an important issue to quantify the cell volume-dependence of the contact areas between the cell and the surrounding ECM. In this study we used two wavelength reflection interference contrast microscopy (DW-RICM) to directly observe the dynamics of cell-substrate contacts, mimicking cell-ECM interactions, in response to a controlled and well-defined volume change induced by hypoosmotic stimulation. This is the first time a non-invasive, label-free method is used to uncover a volume change related response of in vitro hepatocytes in real time. The cell cluster analysis we present here agrees well with previous studies on ex vivo whole liver explants. Moreover, we show that the increase in contact area after cell swelling is a reversible process, while the reorganisation of contacts depends on the type of ECM molecules presented to the cells. As our method complements common whole liver studies providing additional insight on a cell cluster level, we expect this technique to be particular suitable for further detailed studies of osmotic stimulation not only in hepatocytes, but also other cell types.