Verizon LTE 11:30 PM * 56%) instructure-uploads.s3.amazonaws.com Figure The SPB
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Verizon LTE 11:30 PM * 56%) instructure-uploads.s3.amazonaws.com Figure The SPB inheritance network comprises the yeast kinase Swel (also known as Weel), the kinase Kin3 (also known as Nek2) and the acetyltransferase complex NuA4 (also known as Tip60) to control SPB specification. (a) Representative images of cells in meta- and anaphase carrying the SPB-age marker (Spe42-mCherry pre-existing SPB with matured mCherry is brigh new SP with not fully matured mCherry is dim) and an SPB marker (Spe72-sGFP) The arrowhead marks the new SPB (b) Quantification of anaphase cells of the indicated genotype segregating the new SPB into the budO6)(n . 3 independent experiments, except wild type (NT)sve! and so! kisayaf9'.4, with a total of >120 cells per genotype analysed) (c) Categorization of Ka9-YFP asymmetry, Kar9 towards one SP weak asymmetry, Kar9 on both SPBs but asymmetric: 'symmetry, about equal amounts of Kar9 on both SPBs in metaphase cells of the indicated genotype (n 3 independent experiments with a total of > 20 cells per genotype analysed. The arrowhead marks Kar9. (d) Measurement of angle()between the spindle axis (CFP Tubl) and mother-bod axis in metaphase cells of the indicated cells pooled from 3 independent experiments The central mark is the median, puas ()marks the mean, the upper whisker is the maximum and lower whisker is the minimum (e) Quantification of metaphase cells of the indicated genotype with Kar9-YFP on the new SPB (dim Spe4z-m/Cherry) and anaphase cells with the new SPB segregated into the bud()-3 independent experiments, except WT, sipel& and nudl-44n-4, with a total of >120 cells per genotype analysed). The arrowhead marks the new SPB For all panels. all statistical significances were calculated using one-way ANOVA m? 0.0001"P001,P0.0S NS, nos significant. All eror bars represent mean : sd. Scale bars, 2 pm Source data for panels b-e are available in Supplementary Table L Supplementary Figure1 Inactivation of modifying enzymes that localise to or post-translationally modify SPBs or centrosomes and analysis of effect on SPB inheritance.Explanation / Answer
a. Spc72 is the marker protein of SPB (Spindle Pole Body). To monitor the localization of SPB, here Spc72 is tagged with the ‘superfold’ GFP. Spc42, which is tagged with mCherry, is a marker of the SPB age. Thus, in case of the pre-existing SPB of mother cells, Spc42-mCherry was appeared as a brighter puncta compared to the newly formed SPB of the budding cell in both metaphase and anaphase stage. This indicates that pre-existing SPB contains more mature Spc-42, compared to the newly formed SPB of buds that contains immature Spc42. Here, arrowhead indicates the position of new SPB.
b. In this experiment, the authors investigated the effect of the enzymes, which are reported to modulate SPBs post translationally, or they localize on the SPBs. These enzymes are inactivated, and investigate how their inactivation affects the SPB inheritance. In this experiment they measure the number of anaphase cells of the indicated genotype segregating the new SPB into the bud. They used different mutant including the Wee1 kinase Swe1, the Nek2 kinase Kin3 and the acetyltransferase NuA4/Tip60. A larger fraction of swe1 and kin3 single-mutant and NuA4-defective cells (yaf91, eaf11, esa1-414) segregated the new SPB into the bud compared with wild-type cells. Rapid depletion of either Swe1 or Kin3 using the auxin-inducible degron (SWE1-AID and KIN3-AID) increased SPB mis-segregation more than twofold. Swe1 phosphorylates and inhibits Cdc28 (Cdk1) in complex with the cyclins Clb1. Surprisingly, none of the CDC28-Y19F and CDC28-T18AY19F mutation, which withstand Swe1 inhibition, and individual deletions of CLB1_4 affected SPB inheritance. Therefore, Swe1 regulates SPB inheritance through other targets. The swe1 kin3 double mutant segregated SPBs like the single-mutant cells. Thus, Swe1 and Kin3 function in one pathway. In contrast, deleting YAF9 together with SWE1 or KIN3 further randomized SPB segregation. Hence, NuA4 acts in parallel to Swe1 and Kin3. The swe1 kin3 yaf91 mutant cells missegregate SPBs most frequently. Therefore, Swe1, NuA4 and Kin3 form a nonlinear network, which regulates the age-dependent inheritance of SPBs
c. In this experiment they examine that these above-mentioned mutants properly establish the asymmetry of Kar9, on which the proper orientation of spindle depends. They categorized the Kar9-YFP as:‘asymmetry’, Kar9 towards one SPB; ‘weak asymmetry’, Kar9 on both SPBs but asymmetric; ‘symmetry’, about equal amounts of Kar9 on both SPBs in metaphase cells of the indicated genotype. Unlike the positive control (nud1-44), the swe1, Swe1-depleted, kin3, yaf9 and swe1 kin3 1yaf9 mutant cells localized Kar9-YFP as asymmetrically as wild-type cells.
d. They measure the angle between the spindle axis (CFPTub1) and mother–bud axis in metaphase cells of the indicated genotype. Results indicate that above-mentioned mutant cells also align their mitotic spindle with the mother bud axis.
e. They performed quantification of metaphase cells of the indicated genotype with Kar9-YFP on the new SPB (dim Spc42-mCherry) and anaphase cells with the new SPB segregated into the bud. However, many of them pointed Kar9 asymmetry towards the new instead of the pre-existing SPB. Thus, all these mutants do not promote Kar9 asymmetry, but orients it towards the pre-existing SPB.
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