Fission yeast orb6, a ser/thr protein kinase related to mammalian rho kinase and myotonic dystrophy kinase, is required for maintenance of cell polarity and coordinates cell morphogenesis with the cell cycle

Abstract

The molecular mechanisms that coordinate cell morphogenesis with the cell cycle remain largely unknown. We have investigated this process in fission yeast where changes in polarized cell growth are coupled with cell cycle progression. The orb6 gene is required during interphase to maintain cell polarity and encodes a serine/threonine protein kinase, belonging to the myotonic dystrophy kinase/cot1/warts family. A decrease in Orb6 protein levels leads to loss of polarized cell shape and to mitotic advance, whereas an increase in Orb6 levels maintains polarized growth and delays mitosis by affecting the p34(cdc2) mitotic kinase. Thus the Orb6 protein kinase coordinates maintenance of cell polarity during interphase with the onset of mitosis. orb6 interacts genetically with orb2, which encodes the Pak1/Shk1 protein kinase, a component of the Ras1 and Cdc42-dependent signaling pathway. Our results suggest that Orb6 may act downstream of Pak1/Shk1, forming part of a pathway coordinating cell morphogenesis with progression through the cell cycle.

Figure 1

Cloning of orb6⁺. (a) leu1–32, pRep3X. (b) orb6–25 leu1–32, pRep3X. (c) orb6–25 leu1–32, pRep3X orb6⁺. Cells were exponentially grown at 25°C for at least eight generations, then shifted at 36°C for 5 hr.

Figure 2

The orb6⁺ gene. (A) Sequence comparison of Orb6 and homologous proteins. An homology search using EMBL and GenBank databases identified S. cerevisiae 1050791, Neurospora crassa cot1 (14), human Ndr (23), Drosophila warts (15, 16), human PK428, Rho-associated kinase (–19), and human DMPK (20). (B) orb6⁺ gene deletion. Spores were plated on yeast extract plates and allowed to germinate for 16 hr at 32°C. (a) ade- leu1–32 ura4-D18 microcolony. (b) orb6∷ura4⁺ ade- leu1–32 ura4-D18 microcolony. (c) orb6∷ura4⁺ ade- leu1–32 ura4-D18 microcolony after 24 hr at 32°C. (Bar = 5 μm.)

Figure 3

Orb6⁺ gene dosage effects. orb6∷ura4⁺ ade- leu1–32 ura4-D18 cells containing integrated HA-tagged orb6⁺ were grown exponentially for at least eight generations at 32°C, then thiamine was added to the culture. (a and d) Cells grown in the absence of thiamine. (b and e) 7.5 hr after thiamine addition. (c and f) 12 hr after thiamine addition. Cells were photographed immediately (a–c) or fixed and stained for actin (d–f). (Bar, a–c = 5 μm.; d–f = 10 μm.)

Figure 4

Effects of orb6 overexpression. (a–c) Overexpression of HA-tagged orb6⁺ in a leu1–32 strain (experiment 1 in Table 1). (a) leu1–23 pRep3X. (b) leu1–23 cells, carrying integrated HA-tagged orb6⁺, were grown exponentially for at least eight generations at 32°C in the absence of thiamine. (c) leu1–32 cells, carrying HA-tagged orb6⁺ on the multicopy plasmid pRep3X, were grown in the absence of thiamine for 18 hr at 32°C. (d–f) Overexpression of HA-tagged orb6⁺ in a wee1–50 mutant strain. Cells were grown exponentially at 25°C in the presence of thiamine for at least eight generations; thiamine was washed away and cells were grown in the absence of thiamine at 25°C for 16 hr, then shifted at 36°C for 5 hr. (d) leu1–32, pRep3X orb6HA. (e) wee1–50 leu1–32 pRep3X. (f) wee1–50 leu1–32 pRep3X orb6HA. (g) Western blot comparing the levels of expression of orb6-HA in the experiments described above, as compared with the control tubulin: lane 1, leu1–32 with an integrated copy of orb6-HA; lane 2, leu1–23 pRep3X; lane3, leu1–32 pRep3x orb6 HA; lane 4, leu1–32 pRep3x orb6 K122A HA; lane 5, wee1–50 leu1–32 pRep3x; lane 6, wee1–50 leu1–32 pRep3x orb6-HA. (Bar = 5 μm.)

Figure 5

Overexpression of Wee1 in orb6 mutants and actin localization in orb6–25 and wee1–50 mutants. (a–d) Overexpression of Wee1 in orb6 mutants. (a) leu1–32h⁻ pRep3x. (b) orb6–25 leu1–32h⁻ pRep3x. (c) leu1–32h⁻ pRep3x wee1+. (d) orb6–25 leu1–32h⁻ pRep3x wee1+. (e) wee1–50h⁻. Cells were grown exponentially at 25°C in the presence of thiamine for at least eight generations; thiamine was washed away, and cells were grown in the absence of thiamine at 25°C for 16 hr, then shifted at 36°C for 5 hr. (f–h) Actin localization in orb6–25 and wee1–50 mutants. (f) leu1–32h⁻. (g) orb6–25 leu1–32h⁻. (h) wee1–50h⁻. (Bar = 5 μm.)

Figure 6

orb6⁺ gene product intracellular localization in the cell cycle. (a, b, e, and f) orb6∷ura4⁺ ade- leu1–32 ura4-D18 cells containing integrated HA-tagged orb6⁺ were grown exponentially at 32°C in the absence of thiamine, then fixed and stained with an antibody recognizing the HA tag (a and b) and with 4′,6-diamidino-2-phenylindole (e and f). (g) Control staining of orb6∷ura4⁺ ade- leu1–32 ura4-D18 cells containing integrated untagged orb6⁺. (c) cdc25–22 leu1–32 Rep3X orb6 HA cells were grown exponentially at 25°C in the presence of thiamine for at least eight generations; thiamine was washed away and cells were grown in the absence of thiamine at 25°C for 16 hr, then shifted at 36°C for 4 hr. (d) orb2–34 leu1–32 pRep3X orb6 HA at 32°C; cells were stained 14 hr after thiamine depletion, which is about 2 hr after derepression of the nmt1 promoter. (h) orb2–34 leu1–32 stained for actin at 32°C. (Bar = 10 μm.)

Figure 7

Cloning of the orb2⁺ gene. (a) top, ade6-M210 leu1–32h⁻; left, orb6–25 ade6-M210 leu1–32h⁻; bottom, orb2–34 ade6-M210 leu1–32h⁻; right, orb2–34 orb6–25 ade6-M210 leu1–32 h⁻. Cells were grown at 32°C. (b) ade6-M210 leu1–32h⁻, Rep3X. (c) orb2–34 ade6-M210 leu1–32h⁻, pRep3X. (d) orb2–34 ade6-M210 leu1–32h⁻ pDB248′-8 (genomic fragment). (e) orb2–34 ade6-M210 leu1–32h⁻ pRep3X pak1/shk1⁺. (f) orb2–34 ade6-M210 leu1–32h⁻ pRep3X orb6⁺. Cells were grown exponentially at 25°C in the presence of thiamine for at least eight generations; thiamine was washed away and cells were grown in the absence of thiamine at 25°C for 16 hr, then shifted at 36°C for 6 hr. (Bar = 5 μm.)

Figure 8

Orb6 kinase is proposed to be part of a regulatory cascade contributing to the mitotic control and responding to the Ras1 and Cdc42-dependent morphological control pathway via the Pak1/Shk1 kinase. Orb6 kinase regulates independently cell morphogenesis and the cell cycle, mediating its effect on p34^cdc2 kinase directly through the Wee1 protein kinase or through some other mechanism dependent on p34^cdc2 tyrosine phosphorylation.