Mitotic spindle formation in the absence of Polo kinase.
Mitosis is a fundamental process in every eukaryote, in which chromosomes are segregated into two daughter cells by the action of the microtubule (MT)-based spindle. Despite this common principle, genes essential for mitosis are variable among organisms. This indicates that the loss of essential genes or bypass of essentiality (BOE) occurred multiple times during evolution. While many BOE relationships have been recently revealed experimentally, the bypass of essentiality of mitosis regulators (BOE-M) has been scarcely reported, and how this occurs remains largely unknown. Here, by mutagenesis and subsequent evolutionary repair experiments, we isolated viable fission yeast strains that lacked the entire coding region of Polo-like kinase (Plk), a versatile essential mitotic kinase. The BOE of Plk was enabled by specific mutations in the downstream machinery, including the MT-nucleating γ-tubulin complex, and more surprisingly, through down-regulation of glucose uptake, which is not readily connected to mitosis. The latter bypass was dependent on casein kinase I (CK1), which has not been considered as a major mitotic regulator. Our genetic and phenotypic data suggest that CK1 constitutes an alternative mechanism of MT nucleation, which is normally dominated by Plk. A similar relationship was observed in a human colon cancer cell line. Thus, our study shows that BOE-M can be achieved by simple genetic or environmental changes, consistent with the occurrence of BOE-M during evolution. Furthermore, the identification of BOE-M constitutes a powerful means to uncover a hitherto understudied mechanism driving mitosis and also hints at the limitations and solutions for selecting chemotherapeutic compounds targeting mitosis.
Intro
To find suppressors of essential genes, they make heterozygous deletion in a diploid, sporulate, irradiate the spores with UV, grow the spores in the selective medium for the deletion resistance marker, sequence the colonies obtained.
Results
Fig. 1
[x] plo1D: inviable
[x] plo1D ght5D: viable
[x] slow growth low severity
[x] plo1D viable 0.08% glucose
[x] Slow growth high severity No ctrl
[x] plo1 and:
[x] git1: low sev
[x] pka1: low sev
[x] alp4-D440E: low sev
[x] alp6-v664f: high sev
[x] alp4-D440E git1: lower even severity
[x] alp6-v664f git1: lower even
[x] asp1-D507G git1: normal
[x] Others? I guess not since they are not complete deletions, they can be reduced to the genetic interactions.
Fig. 2
[x] plo1D
[x] monopolar spindle
[x] normal nuclear insertion
[x] reduced levels of alp6 on the spb
[x] cut7D monopolar spindle
[x] higher levels of alp6 on the spb
[x] cdc31 monopolar spindle
[x] higher levels of alp6 on the spb
Fig. 3
[x] include mutants
[x] alp4-D440E plo1D:
[x] 30% monopolar spindles
[x] asp1-D507G plo1Δ alive
Fig. 4
[x] ght5D
[x] interphase mts persist during mitosis
[x] resistance to TBZ
[x] normal recruitment of mid1 to cytokinetic ring
[x] ght5D plo1D
[x] mislocalized septum
[x] reduced recruitment of mid1 to cytokinetic ring
[x] No localization of cdc7 to the spb during metaphase
PMID:35286199
Mitotic spindle formation in the absence of Polo kinase.
Mitosis is a fundamental process in every eukaryote, in which chromosomes are segregated into two daughter cells by the action of the microtubule (MT)-based spindle. Despite this common principle, genes essential for mitosis are variable among organisms. This indicates that the loss of essential genes or bypass of essentiality (BOE) occurred multiple times during evolution. While many BOE relationships have been recently revealed experimentally, the bypass of essentiality of mitosis regulators (BOE-M) has been scarcely reported, and how this occurs remains largely unknown. Here, by mutagenesis and subsequent evolutionary repair experiments, we isolated viable fission yeast strains that lacked the entire coding region of Polo-like kinase (Plk), a versatile essential mitotic kinase. The BOE of Plk was enabled by specific mutations in the downstream machinery, including the MT-nucleating γ-tubulin complex, and more surprisingly, through down-regulation of glucose uptake, which is not readily connected to mitosis. The latter bypass was dependent on casein kinase I (CK1), which has not been considered as a major mitotic regulator. Our genetic and phenotypic data suggest that CK1 constitutes an alternative mechanism of MT nucleation, which is normally dominated by Plk. A similar relationship was observed in a human colon cancer cell line. Thus, our study shows that BOE-M can be achieved by simple genetic or environmental changes, consistent with the occurrence of BOE-M during evolution. Furthermore, the identification of BOE-M constitutes a powerful means to uncover a hitherto understudied mechanism driving mitosis and also hints at the limitations and solutions for selecting chemotherapeutic compounds targeting mitosis.
Intro
To find suppressors of essential genes, they make heterozygous deletion in a diploid, sporulate, irradiate the spores with UV, grow the spores in the selective medium for the deletion resistance marker, sequence the colonies obtained.
Results
Fig. 1
[x] plo1D: inviable
[x] plo1D ght5D: viable
[x] plo1D viable 0.08% glucose
Slow growth high severityNo ctrl[x] plo1 and:
Fig. 2
normal nuclear insertioncut7D monopolar spindlehigher levels of alp6 on the spbcdc31 monopolar spindlehigher levels of alp6 on the spbFig. 3
Fig. 4
[x] ght5D
[x] ght5D plo1D
Fig. 5
[x] Synthetic lethal in ght5D plo1D: