Journal: bioRxiv

Article Title: Rap1 binding and a lipid-dependent helix in talin F1 domain promote integrin activation in tandem

doi: 10.1101/504894

Figure Lengend Snippet: (A) Airyscan LSM Image MIP projections of 3T3 fibroblasts expressing both THD-EGFP (green) and mRby2-Paxillin (red). Bottom panel shows the merged image and the paxillin rich ROIs (red lines) with quantitative colocalization of both THD to paxillin in peripherally labelled paxillin rich focal adhesions. The images clearly show a diminishment of co-localized signal of THD to paxillin between the WT control and THD(R35E) mutant, which is further decreased in the THD(R118E) mutant. Quantitation results are displayed in (B) . The number of analyzed adhesions is indicated for each condition. (C) A model of talin1 F1 domain in the cytosol with a positively charged unstructured loop (green) and membrane-associated Rap1b-GTP by its C-terminal geranyl-geranyl (G-G) moiety (orange). Negatively charged phospholipids PI(4,5)P 2 are colored in red. On proximity of the plasma membrane, the low-affinity talin1 F1 Ras-associating (RA) domain probes for Rap1 and the F1 loop seeks negatively charged phospholipids. On contact with Rap1 and negatively charged phospholipids, the F1 RA domain interacts with Rap1 and the F1 loop helical state is favored resulting in cluster of positive charges on one side of the helix. View of THD (green) as seen from the membrane is displayed in the top right panel. Bottom panel shows view of THD as seen from the side. The views highlight the position of the THD F0, F1, F2 and F3 subdomains and the Rap1b (orange) bound to the F0 and F1 subdomains. Both Rap1b C-terminal geranyl-geranyl (G-G) moieties are pointing towards the membrane, as for the F1 loop, the F2 membrane orientation patch (MOP), the F3 association patch (FAP), and the position of the F3 β-integrin. The regions known to interact with the negatively charged phospholipids are shown in blue; the F1 fly-casting loop is shown as a helix, the F2 membrane orientation patch (MOP), and the F3 association patch (FAP). The β-integrin transmembrane and cytoplasmic tail is shown in red bound to the F3 subdomain. Two Rap1b (orange) molecules are shown with their C-terminal geranyl-geranyl (G-G) moiety inserted in the membrane. One Rap1b is bound to the F0 and one to the F1 subdomain. This model summarize the multiple known interactions of THD at the plasma membrane; the negatively charged PI(4,5)P 2 , Rap1 and integrin β-tails.

Article Snippet: Each image consisted of z-stacks of multiple frames that were first processed from a raw Airyscan image to a final integrated, corrected and deconvolved image that was then flattened as a maximum intensity projection using the ZEN software (Zeiss Inc.).

Techniques: Expressing, Control, Mutagenesis, Quantitation Assay, Membrane, Clinical Proteomics

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging

doi: 10.1007/978-1-0716-1402-0_5

Figure Lengend Snippet: Airyscan detector in Airyscan mode (A) and Airyscan FAST mode (B). (A) The Airyscan detector consists of 32 hexagonal elements arranged in a circular disk. Each detector element acts as a pinhole the size of 0.2 Airy Units (AU). The whole detector captures the light equivalent to a pinhole of 1.25 AU. As the sample is scanned by point spot illumination from the laser, the Airy disk arising from a point emitter will be scanned by the detector. Through deconvolution and pixel reassignment, the individual images from each detector element are shifted to the center position to yield a final super-resolution image with resolutions of 120 nm in x and y and 350 nm in z. (B) In the FAST mode, the illumination is shaped like a short elliptical line. Each detector element has an equivalent pinhole size of 0.3 AU, and the central 16 elements of the detector will capture the light from 0.9 AU in x and 1.6 AU in y. Because the elliptical line scans four spots simultaneously instead of one, it scans the sample four times faster than in the regular Airyscan mode.

Article Snippet: Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging

doi: 10.1007/978-1-0716-1402-0_5

Figure Lengend Snippet: Common configurations for Airyscan SR imaging. Select these components in the “Imaging Setup” window. The options for each filter set are listed in .

Article Snippet: Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope.

Techniques: Imaging

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging

doi: 10.1007/978-1-0716-1402-0_5

Figure Lengend Snippet: The screen shot indicating good alignment of the Airyscan detector.

Article Snippet: Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging

doi: 10.1007/978-1-0716-1402-0_5

Figure Lengend Snippet: Key screen shots (left) and quick guides (right) for Airyscan Acquisition. (A) “Channel” window. (B) “Acquisition Mode” window.

Article Snippet: Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope.

Techniques:

Journal: Methods in molecular biology (Clifton, N.J.)

Article Title: ZEISS Airyscan: Optimizing usage for fast, gentle, super-resolution imaging

doi: 10.1007/978-1-0716-1402-0_5

Figure Lengend Snippet: Two-color, line switch Airyscan scan time at a given zoom. The scan time listed is under the following conditions: no averaging (ave 1), bi-directional scan, and at 8-bit depth.

Article Snippet: Of note, while our focus is on the Airyscan function of this microscope rather than its conventional confocal function, the Airyscan unit comes as an add-on to the conventional Zeiss laser scanning confocal microscope.

Techniques: