Journal: Cell Reports

Article Title: Phosphatidic acid-dependent recruitment of microtubule motors to spherical supported lipid bilayers for in vitro motility assays

doi: 10.1016/j.celrep.2024.114252

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Article Snippet: 5 micron syringe filter , Sartorius , 16517E.

Techniques: Recombinant, Protease Inhibitor, Liquid Chromatography with Mass Spectroscopy, Software

Journal: The Journal of Cell Biology

Article Title: A Cell-Free Assay Allows Reconstitution of Vps33p-Dependent Transport to the Yeast Vacuole/Lysosome

doi:

Figure Lengend Snippet: Lysis of yeast spheroplasts via extrusion through a polycarbonate filter. (A) Schematic diagram for polycarbonate filter lysis and subsequent differential centrifugation. After pushing a yeast spheroplast suspension in a syringe through a polycarbonate filter with 3-μm pores, the crude cell lysate was subjected to differential centrifugation using the indicated g forces and times. The P1, P2, and P3 pellets were enriched in the indicated organelles. (B) Fractionation of vacuolar marker proteins. Wild-type yeast spheroplasts (SEY6210) were radiolabeled with Tran 35 S-label for 5 min and chased with methionine and cysteine for 2 min at 30°C. The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation. Each supernatant and pellet from the 440 (S1 and P1), 15,000 (S2 and P2), and 125,000 g (S3 and P3) centrifugation steps was sequentially immunoprecipitated with antisera against carboxypeptidase Y (CPY), proteinase A (PrA), and alkaline phosphatase (ALP). Each immunoprecipitate was subjected to SDS-PAGE and autoradiography. The mid and late Golgi complex–modified precursor zymogen is designated p2, the endoplasmic reticulum and early Golgi complex precursor is designated p1 (CPY only), and the mature hydrolase is designated m for each protein.

Article Snippet: The resuspended cells were pushed through a 13-mm polycarbonate filter (NucleoporeTM; Corning) with 3-μm pores using a 3-ml syringe.

Techniques: Lysis, Centrifugation, Suspension, Fractionation, Marker, Immunoprecipitation, SDS Page, Autoradiography, Modification

Journal: The Journal of Cell Biology

Article Title: A Cell-Free Assay Allows Reconstitution of Vps33p-Dependent Transport to the Yeast Vacuole/Lysosome

doi:

Figure Lengend Snippet: Light microscopy of cell-free membrane pellets after polycarbonate filter lysis. Wild-type yeast cells (as in ) were first stained with FM4-64 (15-min pulse, 45-min chase) followed with dichlorocarboxyfluorescein diacetate (15 min at pH 4.0) to mark the vacuole membrane and lumen, respectively. The double-stained cells were then enzymatically converted to spheroplasts at 30 or 15°C (as indicated). The spheroplasts were extruded through a polycarbonate filter with 3-μm pores (as described in A). The lysate, P1, P2, and P3 pellets (as indicated) were then examined under a light microscope with differential interference contrast (DIC), phase contrast, and epifluorescence optics using a FITC and Texas red filter set (as indicated). The fluorescence images were digitally overlaid for a composite. The cells at 15°C (inset) were stained with just FM4-64 for a 30-min pulse. Bar, 5 μm.

Article Snippet: The resuspended cells were pushed through a 13-mm polycarbonate filter (NucleoporeTM; Corning) with 3-μm pores using a 3-ml syringe.

Techniques: Light Microscopy, Membrane, Lysis, Staining, Fluorescence

Journal: The Journal of Cell Biology

Article Title: A Cell-Free Assay Allows Reconstitution of Vps33p-Dependent Transport to the Yeast Vacuole/Lysosome

doi:

Figure Lengend Snippet: Vacuolar precursor proteins undergo maturation after incubating donor and acceptor membranes in a cell-free system. Wild-type yeast spheroplasts were radiolabeled (as in B). The cells were subjected to lysis through a polycarbonate filter with subsequent differential centrifugation to generate a 125,000 g , P3 donor membrane pellet. The same yeast strain was used to make a 15,000 g , P2 acceptor membrane pellet from nonradiolabeled spheroplasts. The radiolabeled donor membranes (from ∼5 × 10 7 spheroplasts per reaction) were incubated at 25°C for 60 min with various combinations of buffer, ATP (plus a regeneration system), cytosol (5 mg/ml), and acceptor membranes (∼100 μg) in a total volume of 50 μl, as indicated. All reactions were sequentially immunoprecipitated for CPY and PrA, subjected to SDS-PAGE, and autoradiography. For the reactions in lanes 10 and 11, both donor and acceptor membranes were washed once with lysis buffer and reharvested before incubation with ATP and cytosol.

Article Snippet: The resuspended cells were pushed through a 13-mm polycarbonate filter (NucleoporeTM; Corning) with 3-μm pores using a 3-ml syringe.

Techniques: Lysis, Centrifugation, Membrane, Incubation, Immunoprecipitation, SDS Page, Autoradiography