Technical Reference #3149

3149.

Elm1 kinase activates the spindle position checkpoint kinase Kin4 Ayse Koca Caydasi; Bahtiyar Kurtulmus; Maria I.L. Orrico; Astrid Hofmann; Bashar Ibrahim; and Gislene Pereira, German Cancer Research Center, Journal of Cell Biology, 190(3149), (2010)
Link To Paper

Abstract:
Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

Materials & Methods:
Fluorescence microscopy Time-lapse and FRAP experiments were performed on glass-bottom dishes (MatTek) using 6% concanavalin A-Type IV (Sigma-Aldrich) for cell adhesion. Images were acquired at 30°C using a wide-field fluorescence imaging system (DeltaVision RT; Applied Precision) equipped with a 100×/1.40 NA UPLS Apochromat UIS2 oil immersion objective lens a charge-coupled device camera (CoolSNAP HQ/ICX285; Photometrics) a quantifiable laser module and SoftWoRx software (Applied Precision). Nineteen z stacks of 0.2 μm thickness were taken for Kin4-GFP (3-min intervals) and Elm1-GFP (every minute). For GFP-tubulin images 12 z stacks of 0.3 μm thickness were taken every minute. FRAP of Elm1-GFP was performed by bleaching 80–100% of the Elm1-GFP at the bud neck with two laser (20 mW 488 nm laser) iterations of 0.01 s duration (50% intensity) each and by acquiring single plane pre- and postbleach images. The mean signal intensity at the entire bud neck region was quantified using ImageJ (National Institutes of Health) software corrected for acquisition bleaching and normalized as described in Caydasi and Pereira (2009). FRAP recovery curves were fitted to a single exponential curve (y = yo + Ae  bx) using IgorPro 6.02 (WaveMetrics) software. Half recovery times were calculated as –ln0.5b. Time-lapse images of Elm1-GFP and Kin4-GFP shown in Fig. S4 (A and B) were deconvolved and z-projected using SoftWoRx. In other figures z stacks were projected without deconvolution using ImageJ software.

Microscopic Technique
Fluorescence microscopy, Time-lapse and FRAP experiments

Cell Type(s)
Yeast