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First published online 9 November 2004
doi: 10.1242/jcs.01525

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Scribble protein domain mapping reveals a multistep localization mechanism and domains necessary for establishing cortical polarity

Roger Albertson^*, Chiswili Chabu, Amy Sheehan and Chris Q. Doe

Institute of Molecular Biology, Institute of Neuroscience, Howard Hughes Medical Institute, University of Oregon 1254, Eugene, OR 97403, USA

View larger version (29K): [in a new window]   Fig. 1. Generation and characterization Scrib domain constructs. (A) Schematic representation of the seven different Scrib:myc proteins used in this study. LRR repeats (green ovals), LAPSDa (red oval), LAPSDb (red square), PDZ domains (blue boxes) and C-terminal region (red line) are indicated. FL is the full-length Scrib; CT lacks the C-terminal region; LRR contains only the N-terminal LRR region; PDZ lacks the central PDZ region; PDZ contains only the central PDZ region; LRR lacks the N-terminal LRR region; CT contains only the C-terminal region. The numbers above each line represent the amino acids present at the beginning or end of each domain. All constructs carry six myc tags (white circle). (B) Western blot showing the size and stability of Scrib:myc proteins in vivo. Embryos expressing each Scrib:myc proteins (progeny of Sca-Gal4 x UAS-scrib:myc crosses) were homogenized, run in the indicated lanes and the western blot was probed with a Myc antibody.

View larger version (44K): [in a new window]   Fig. 2. Scrib domain localization in scrib mutant neuroblasts and epithelia of stage 15 embryos. Left column, metaphase neuroblasts; middle column, interphase neuroblasts; right column, interphase epithelial cells; apical up for all images. The genotype is indicated to the left of each row; antibodies used to detect endogenous Scrib (A) or Scrib:myc (B-H) are indicated to the right of each row. (A) Wild-type embryos showing endogenous Scrib protein localization. (B-H) scrib1/scrib1 embryos showing localization of the indicated Scrib:myc proteins detected with Myc antibody. Each UAS-scrib:myc transgene was expressed using worniu-gal4 (for the neuroblasts) or scabrous-gal4 (for the epithelial). Insets show the same neuroblast stained for -tubulin (green) as a cell cycle indicator and Scrib (A) or Scrib:myc (B-H) protein (red). Scrib domain abbreviations are given in Fig. 1. (I) Quantification of endogenous Scrib and Scrib domain localization in stage 15 metaphase neuroblasts. Apical enrichment, black bar; uniform cortical localization, crosshatched red bar; cytoplasmic localization, blue bar. Scrib domain abbreviations are given in Fig. 1.

View larger version (43K): [in a new window]   Fig. 3. Scrib domain regulation of Mira basal localization in stage 15 neuroblasts. Top row, Mira localization in metaphase neuroblasts; middle row, the same neuroblasts showing both Mira (green) and the mitotic DNA marker phosphohistone H3 (red); lower row, quantification of the Mira localization phenotype: normal basal crescent, black bar; uniform cortical localization, crosshatched bar; uniform cortical plus cytoplasmic and spindle-associated, blue bar; apical up for all images. Scrib domain abbreviations are given in Fig. 1. (A) Wild-type neuroblast showing normal Mira basal crescent. (B) scrib1/scrib1 neuroblast showing cytoplasmic and spindle-associated Mira. (C-I) scrib1/scrib1 embryos expressing the indicated Scrib:myc transgene (progeny from UAS-scrib:myc x worniu-gal4). (C,D) FL and CT proteins provide full rescue of basal Mira localization. (E,F) LRR and PDZ result in predominantly uniform cortical Mira localization or cortical with weak basal enrichment. (G-I) PDZ, LRR and CT proteins give no rescue of Mira cortical or basal localization.

View larger version (49K): [in a new window]   Fig. 4. Scrib domain regulation of apical protein localization in stage 15 neuroblasts. Embryonic neuroblasts stained for the apical proteins aPKC or Dlg (green) and mitotic markers -tubulin or PhosphohistoneH3 (PH3; red). Stage 15 embryos, apical up for all images. Scrib domain abbreviations given in Fig. 1. (A) Wild-type metaphase neuroblast showing apical localization of aPKC and Dlg. (B-H) scrib1/scrib1 metaphase neuroblasts expressing the indicated Scrib:myc transgene (progeny from UAS-scrib:myc x worniu-gal4). All show normal localization of aPKC and Dlg.

View larger version (24K): [in a new window]   Fig. 5. Scrib domain regulation of mitotic spindle and cell size asymmetry in neuroblasts. Stage 15 telophase neuroblasts (identified as Pins+ and Mira+) were scored for cortical markers, spindle morphology, and cell size. Schematic of the phenotypic categories is shown at the top: apical cortex, blue (Pins); basal cortex, red (Mira); mitotic spindle and centrosomes, green (-tubulin). Cell size ratios are apical/basal and are defined as: normal, >1.25; symmetrical, 0.8-1.25; inverted, <0.8. Wild-type neuroblasts divide asymmetrically. scrib1/scrib1 neuroblasts show only 65% asymmetric divisions. scrib1/scrib1 neuroblasts expressing FL or CT proteins rescue normal asymmetric divisions. scrib1/scrib1 neuroblasts expressing LRR, PDZ, PDZ, LRR, or CT proteins fail to rescue asymmetric cell division.

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