Background: ER stress induces cell surface translocation of GRP78/BiP. Results: GRP78 translocation to the cell surface requires its substrate binding domain and exists majorly as a peripheral protein.Conclusion: GRP78 anchors on the cell surface via interaction with other proteins, and the translocation mechanism is cell context-dependent. Significance: Learning how GRP78 exists on the cell surface is crucial for understanding its signaling regulatory functions.

Single-molecule (SM) fluorescence microscopy allows the imaging of biomolecules in cultured cells with a precision of a few nanometres but has yet to be implemented in living adult animals. Here we used split-GFP (green fluorescent protein) fusions and complementation-activated light microscopy (CALM) for subresolution imaging of individual membrane proteins in live Caenorhabditis elegans (C. elegans). In vivo tissue-specific SM tracking of transmembrane CD4 and voltage-dependent Ca2+ channels (VDCC) was achieved with a precision of 30 nm within neuromuscular synapses and at the surface of muscle cells in normal and dystrophin-mutant worms. Through diffusion analyses, we reveal that dystrophin is involved in modulating the confinement of VDCC within sarcolemmal membrane nanodomains in response to varying tonus of C. elegans body-wall muscles. CALM expands the applications of SM imaging techniques beyond the petri dish and opens the possibility to explore the molecular basis of homeostatic and pathological cellular processes with subresolution precision, directly in live animals.

Patterning cells on microcontact-printed substrates is a powerful approach to control cell morphology and introduce specific mechanical cues on a cell's molecular organization. Although global changes in cellular architectures caused by micropatterns can easily be probed with diffraction-limited optical microscopy, studying molecular reorganizations at the nanoscale demands micropatterned substrates that accommodate the optical requirements of single molecule microscopy techniques. Here, we developed a simple micropatterning strategy that provides control of cellular architectures and is optimized for nanometer accuracy single molecule tracking and three-dimensional super-resolution imaging of plasma and nuclear membrane proteins in cells. This approach, based on fibronectin microcontact printing on hydrophobic organosilane monolayers, allows evanescent wave and light-sheet microscopy of cells whilst fulfilling the stringent optical demands of point reconstruction optical microscopy. By imposing steady-state mechanical cues on cells grown in these micropatterns, we reveal nanoscale remodeling in the dynamics and the structural organizations of the nuclear envelope mechanotransducing protein emerin and of the plasma membrane mechanosensing protein caveolin-1 using single particle tracking photoactivated localization microscopy and direct stochastic optical reconstruction microscopy imaging. In addition to allowing quantitative biophysical studies of mechanoresponsive membrane proteins, this approach provides an easy means to probe mechanical regulations in cellular membranes with high optical resolution and nanometer precision.

Mutagenic translesion DNA polymerase V (UmuD′ 2 C) is induced as part of the DNA damage-induced SOS response in Escherichia coli , and is subjected to multiple levels of regulation. The UmuC subunit is sequestered on the cell membrane (spatial regulation) and enters the cytosol after forming a UmuD′ 2 C complex, ~ 45 min post-SOS induction (temporal regulation). However, DNA binding and synthesis cannot occur until pol V interacts with a RecA nucleoprotein filament (RecA*) and ATP to form a mutasome complex, pol V Mut = UmuD′ 2 C-RecA-ATP. The location of RecA relative to UmuC determines whether pol V Mut is catalytically on or off (conformational regulation). Here, we present three interrelated experiments to address the biochemical basis of conformational regulation. We first investigate dynamic deactivation during DNA synthesis and static deactivation in the absence of DNA synthesis. Single-molecule (sm) TIRF-FRET microscopy is then used to explore multiple aspects of pol V Mut dynamics. Binding of ATP/ATPγS triggers a conformational switch that reorients RecA relative to UmuC to activate pol V Mut. This process is required for polymerase-DNA binding and synthesis. Both dynamic and static deactivation processes are governed by temperature and time, in which on → off switching is “rapid” at 37°C (~ 1 to 1.5 h), “slow” at 30°C (~ 3 to 4 h) and does not require ATP hydrolysis. Pol V Mut retains RecA in activated and deactivated states, but binding to primer-template (p/t) DNA occurs only when activated. Studies are performed with two forms of the polymerase, pol V Mut-RecA wt, and the constitutively induced and hypermutagenic pol V Mut-RecA E38K/ΔC17. We discuss conformational regulation of pol V Mut, determined from biochemical analysis in vitro , in relation to the properties of pol V Mut in RecA wild-type and SOS constitutive genetic backgrounds in vivo .

GRP78 conducts protein folding and quality control in the ER and shows elevated expression and cell surface translocation in advanced tumors. However, the underlying mechanisms enabling GRP78 to exert novel signaling functions at cell surface are just emerging. CD44 is a transmembrane protein and an important regulator of cancer metastasis, and isoform switch of CD44 through incorporating additional variable exons to the extracellular juxtamembrane region is frequently observed during cancer progression. Using super-resolution dual-color single-particle tracking, we report that GRP78 interacts with CD44v in plasma membrane nanodomains of breast cancer cells. We further show that targeting cell surface GRP78 by the antibodies can effectively reduce cell surface expression of CD44v and cell spreading of tamoxifen-resistant breast cancer cells. Our results uncover new functions of GRP78 as an interacting partner of CD44v and as a regulator of CD44v membrane homeostasis and cell spreading. This study also provides new insights into anti-CD44 therapy in tamoxifen-resistant breast cancer.