Gandhi, H. | Search bioRxiv

bioRxiv2018

Single molecule localization microscopy with autonomous feedback loops for ultrahigh precision

Single-molecule localization microscopy (SMLM) promises to provide truly molecular scale images of biological specimens1-5. However, mechanical instabilities in the instrument, readout errors and sample drift constitute significant challenges and severely limit both the useable data acquisition length and the localization accuracy of single molecule emitters6. Here, we developed an actively stabilized total internal fluorescence (TIRF) microscope that performs 3D real-time drift corrections and achieves a stability of [≤]1 nm. Self-alignment of the emission light path and corrections of readout errors of the camera automate channel alignment and ensure localization precisions of 1-4 nm in DNA origami structures and cells for different labels. We used Feedback SMLM to measure the separation distance of signaling receptors and phosphatases in T cells. Thus, an improved SMLM enables direct distance measurements between molecules in intact cells on the scale between 1-20 nm, potentially replacing Forster resonance energy transfer (FRET) to quantify molecular interactions7. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

biophysics↗

bioRxiv2018

Rapid Whole Cell Imaging Reveals An APPL1-Dynein Nexus That Regulates Stimulated EGFR Trafficking

Multicellular life processes such as proliferation and differentiation depend on cell surface signaling receptors that bind ligands generally referred to as growth factors. Recently, it has emerged that the endosomal system provides rich signal processing capabilities for responses elicited by these factors [1-3]. At the single cell level, endosomal trafficking becomes a critical component of signal processing, as exemplified by the epidermal growth factor (EGF) receptors of the receptor tyrosine kinase family. EGFRs, once activated by EGF, are robustly trafficked to the phosphatase-enriched peri-nuclear region (PNR), where they are dephosphorylated [4-8]. However, the details of the mechanisms regulating the movements of stimulated EGFR in time and space, i.e., towards the PNR, are not known. What endosomal regulators provide specificity to EGFR? Do modifications to the receptor upon stimulation regulate its trafficking? To understand the events leading to EGFR translocation, and especially the early endosomal dynamics that immediately follow EGFR internalization, requires the real-time, long-term, whole-cell imaging of multiple elements. Here, exploiting the advantages of lattice light-sheet microscopy [9], we show that the binding of EGF by its receptor, EGFR, triggers a transient calcium increase that peaks by 30 s, causing the desorption of APPL1 from pre-existing endosomes within one minute, the rebinding of liberated APPL1 to EGFR within three minutes, and the dynein-dependent translocation of APPL1-EGF-bearing endosomes to the PNR within five minutes. The novel, cell spanning, fast acting network that we reveal integrates a cascade of events dedicated to the cohort movement of activated EGFR receptors. Our findings support the intriguing proposal that certain endosomal pathways have shed some of the stochastic strategies of traditional trafficking, and have evolved behaviors whose predictability is better suited to signaling [10, 11]. Work presented here demonstrates that our whole cell imaging approach can be a powerful tool in revealing critical transient interactions in key cellular processes such as receptor trafficking.

cell biology↗