(F) and (H) show the quantification of (E) and (G), respectively. harboring wild-type plasmid, whereas 5-FOA-LEU selects against the (B) cells. (C and D) Quantification of GFP-Fks1 and Myo2-3Cherry at the bud neck in wild-type ((cells. At the first time point, Abp140-GFP localizes at the bud neck as a ring (white arrowhead) (defined as cells. (F) Actin patches and cables were labeled with rhodamine-phalloidine and inspected in large budded cells (in the presence or absence of and without or with overexpression of (endogenous promoter from a 2 -based plasmid, 2 -2 -2 -cells empty or carrying cells carrying an empty or a high-copy plasmid (2 , ((cells transporting with and without at different cell cycle phases. (F) Immunoblot shows protein levels of GFP-Rho1, GFP-Rho1-G19V, GFP-Rho1-Q68H, GFP-Rho1-C25A, and GFP-Rho1-F35L with and without cells. (H) Quantification of Sec3-3Cherry in wild-type (((cells transporting with and without cells transporting with and without cells. Error bars show the standard deviation. Scale pub: 5 m.(TIF) pbio.1001495.s007.tif (2.5M) GUID:?48497DFE-8DBC-42F7-9ABF-AD9C3D021151 Number S8: Gps1 is not required for PIP2 accumulation in the bud neck. (A) PIP2 (stained with GFP-2xPH) concentrates in the bud neck after (top panel) but not before (lower panel) AMR (Myo1) contraction. (BCD) Quantification of PIP2 in the bud ASP6432 neck of wild-type and cells. Plan representation (B) and storyline profiles (C and D) of PIP2 (GFP-2xPH fluorescence intensity) in the bud neck in wild-type and cells. BN, bud neck region. (ECH) Time-lapse series showing PIP2 (GFP-2xPH) localization during cytokinesis in wild-type (E and F) and (G and H) cells. (F) and (H) display the quantification of (E) and (G), respectively. Level bars: 5 m.(TIF) pbio.1001495.s008.tif (2.0M) GUID:?CEE9A8FC-EE75-4200-BC67-AEC1C7DB2428 Figure S9: Gps1 is not required for Rho1 GEF localization in the bud neck. (A) Growth of serial dilutions of and cells transporting the Rho1 GEFs Tus1-GFP, Rom1-GFP, or Rom2-GFP. Note that Shs1-GBP tethers the GFP fusion protein to the bud neck constitutively. (BCD) Still images for the Rho1 GEFs Tus1-GFP, Rom1-GFP, or Rom2-GFP in wild-type, cells in different cell cycle phases. (E) Time-lapse series are demonstrated for the localization of Rom2-GFP in the bud neck in wild-type, cells. Note that Rom2-GFP gives a dim signal in the bud neck in time-lapse analysis (reddish arrowhead) in both wild-type and cells. Level bars: 5 m.(TIF) pbio.1001495.s009.tif (3.2M) GUID:?C20E3395-91A7-4B50-B9D3-CCE2F221FB45 Number S10: Rho GTPase GAPs and their involvement in the Gps1 pathway. (A) Growth of serial dilutions of cells with and without additional deletion of Rho GTPase GAPs as indicated. (B) Time-lapse series display Lrg1-GFP localization in the cell division site in wild-type and cells. (C) Graph shows the quantification of Lrg1-GFP signals in the bud neck in wild-type ((cells with and are stained with the cell death marker propidium iodide (PI). Level pub: 10 m.(TIF) pbio.1001495.s011.tif (3.0M) GUID:?730C332C-4E11-42BC-9BD1-2D5EB90D7EE1 Number S12: Cdc24 localization in cells. Cdc24 build up in the bud neck (white arrowheads) and adjacent to the bud neck (reddish arrowheads) is definitely depicted. White colored asterisks mark a deceased daughter cell.(TIF) pbio.1001495.s012.tif (4.4M) GUID:?CFED12C1-4CBA-42D2-A49A-E0CCA3667CD5 Figure S13: Cdc42-T35A rescue of cells expressing different mutants from a low-copy plasmid (A) or stably integrated into the genome (B). Protein levels are shown. With the exception of GFP-Cdc42-T35A-D118A, the protein amounts ASP6432 of all mutants are comparable to wild-type GFP-Cdc42. Variations between (A) and (B) may result from different manifestation levels.(TIF) pbio.1001495.s013.tif (1.1M) GUID:?E82B476E-55EA-4B3F-93DA-24348345E92A Number S14: Genetic interaction between cells upon deletion of the indicated Cdc42 effectors.(TIF) pbio.1001495.s014.tif (859K) GUID:?CA7962B6-9734-44DC-B3EB-8D5603950D8B Number ASP6432 S15: Cla4 localization in ((and cells determined by immunoblot. An unspecific Rabbit polyclonal to AGO2 transmission was used like a loading control.(TIF) pbio.1001495.s015.tif (339K) GUID:?4ECD0C00-81EB-4B01-AC64-2A46A0ACEFCA Table S1: Save of cell.(MOV) pbio.1001495.s020.mov (89K) GUID:?3603C241-5F1C-419C-9A4E-9789B6328ACF Video S3: Death of the daughter cell in cell.(MOV) pbio.1001495.s021.mov (108K) GUID:?B8185CDB-8F58-4F87-990D-0C2E26AB489F Video S4: Localization of GFP-Cdc42 in crazy type. Phase contrast (remaining), GFP-Cdc42 localization (right), and AMR contraction (middle, Myo1-3Cherry) are demonstrated inside a representative wild-type cell.(MOV) pbio.1001495.s022.mov (87K) GUID:?F8F91305-B23C-401C-8AC3-45045278EDBF Video S5: Localization of GFP-Cdc42 in cell.(MOV) pbio.1001495.s023.mov (71K) GUID:?2F70739F-F6A8-4FDB-9DE5-AF68FD10C5B3 Abstract The spatiotemporal control of cell polarity is vital for the development of multicellular organisms and for reliable polarity switches during.