The Hippo pathway kinases regulate the subcellular localization of coYki.

(A) The Hippo pathway in Drosophila (yellow text) and mammals (white text). Figure from (Phillips et al., 2022). (B) Conservation of Hippo pathway components in close unicellular relatives of animals. 1The Hippo kinase SARAH domain is absent from putative Hippo kinase orthologs in yeasts, but is present in the early-branching fungus Spizellomyces punctatus and in amoebozoans such as Dictyostelium, indicating SARAH domain loss in some fungal lineages. Data from (Sebe-Pedros et al., 2012). (C) Cells were transiently transfected with the indicated constructs and imaged by confocal microscopy. Scale bar is 5 microns. (D) Intensity of mScarlet-coYki signal in the nucleus and cytoplasm of cells was quantified. Where p values are given, differences between conditions are significant (One-way ANOVA, Tukey’s test, n = 3 with at least 10 cells measured for each independent experiment.) Values are mean ± SEM. Absence of error bars indicates that error is smaller than the plot symbol.

The Hippo pathway kinases do not restrict cell proliferation in Capsaspora.

Cells were grown in adherent culture in 96-well plates (A) or in shaking culture (B) and cell density was measured daily by hemocytometer. (C, D) Cells were inoculated under low-adherence conditions to induce aggregate formation, and 3 days after induction aggregates were incubated with EdU for 4 hours, fixed, and processed to detect EdU labeling. Scale bar is 10 microns. Differences in EdU labeling between WT and mutants were not significant (one-way ANOVA, Dunnett’s test, n = 3 with 3 aggregates measured for each independent experiment). Black bars show mean ± SEM, and red dots show values for individual aggregates.

The Hippo pathway kinases regulate cell morphology and a contractile cell behavior in Capsaspora.

(A) Cells were inoculated into glass-bottom chamber slides, and adherent cells were imaged two days after inoculation. Scale bar is 50 microns. (B) The aspect ratio of cells imaged as described in (A) was measured using ImageJ. The differences between WT and either coHpo -/- or coWts -/- cells are significant (one-way ANOVA, Dunnett’s test, n = 3 with 50 cells measured for each independent experiment). Black bars show mean ± SEM, and red dots show values for 150 individual cells. (C) Cells were prepared as described in (A) and imaged by time lapse microscopy. Scale bar is 10 microns.

Characterization of actomyosin in contractile Capsaspora cells.

(A) Cells were inoculated on glass-bottom chamber slides, and at 2 days after inoculation adherent cells were imaged by time-lapse epifluorescence microscopy. Blue arrowheads indicate ventral F-actin-enriched foci, and yellow arrow indicates the F-actin fiber that connects the foci. Scale bar is 10 microns. (B) Cells were inoculated on glass-bottom chamber slides. Two days after inoculation, DMSO or Blebbistatin at a concentration of 1 μM was added to cells. Cells were the imaged 24 hours after addition of blebbistatin. (C) The aspect ratio of cells prepared as in (B) was measured using ImageJ. For both WT and coHpo -/- cells, the difference between DMSO and blebbistatin treatment measurements are significant (t-test, n = 3 with 50 cells measured for each independent experiment). Black bars indicate mean ± SEM, and red dots indicate measurements for 150 individual cells for each condition. Absence of error bars indicates that error is smaller than the plot symbol.

The Hippo pathway kinases regulate the density of cell packing in Capsaspora aggregates.

(A) Aggregates were induced by inoculating cells in low-adherence conditions, and aggregates were imaged at 5 days after inoculation. Scale bar is 100 microns. (B) the size and circularity of aggregates imaged as in (A) was measured using ImageJ. Black bars indicate mean ± SEM (n = 3 with 10 aggregates measured for each independent experiment), and red dots indicate values for 30 individual aggregates. For aggregate circularity, the difference between WT and coWts -/- aggregates is significant (one-way ANOVA, Dunnett’s test.) (C) Cells expressing either mScarlet or N-myristoylation motif (NMM)-Venus, a marker of the plasma membrane which allows visualization of microvilli, were mixed 1:1 and inoculated into low-adherence conditions to induce aggregation. After 5 days, aggregates were imaged by confocal microscopy. Scale bar is 10 microns. (D) Clonal populations of cells of the indicated genotype stably expressing mScarlet were inoculated into low-adherence conditions, and cell aggregates were imaged 5 days after inoculation by confocal microscopy. Scale bar is 20 microns. (E) The number of cells within a 40 micron2 area from aggregate optical sections imaged as in (D) was measured. Black bars indicate mean ± SEM (n = 3 with 3 aggregates measured for each independent experiment), and red dots indicate values for 9 individual aggregates for each condition. The difference between WT and either coHpo -/- or coWts -/- cells is significant (one-way ANOVA, Dunnett’s test.)

Perturbation of the actomyosin network affects cell packing in multicellular Capsaspora aggregates.

(A) Aggregates were treated with 20 μM blebbistatin or DMSO as a vehicle control for 20 minutes and then imaged by confocal microscopy. Scale bar is 20 microns. (B) Cells per area were quantified from images obtained as in (A). Black bars indicate means ± SEM (n = 3 with 3 aggregates measured for each independent experiment), and red dots indicate values for 9 individual aggregates for each condition. Absence of error bars indicates that error is smaller than the plot symbol. For both WT and coHpo -/- aggregates, the difference between DMSO and blebbistatin treatment is significant (t-test). (C) Aggregates were treated with 5 μg/ml Latrunculin B or ethanol as a vehicle control for 1 hour and then imaged by confocal microscopy. Scale bar is 20 microns. (D) Cells per area were quantified from images obtained as in (C). For both WT and coHpo -/- aggregates, the difference between ethanol and Latrunculin B treatment is significant (n = 3, t-test.)

Expression of a constitutively active form of coYki phenocopies loss of coHpo or coWts in Capsaspora.

(A) Aggregates were imaged 5 days after inoculation. Scale bar is 20 microns. (B) The number of cells within a 40 micron2 area within an aggregate was measured. Black bars indicate mean ± SEM (n = 3 with 3 aggregates measured for each independent experiment), and red dots indicate values for 9 individual aggregates for each condition. The difference between WT and EF1 > coYki 4SA is significant (t-test). (C) Cells were inoculated into glass-bottom chamber slides and imaged 2 days after inoculation. Scale bar is 50 microns. (D) The aspect ratio of cells imaged as in (C) was measured using ImageJ. Black bars show mean ± SEM (n = 3 with 40 cells measured for each independent experiment), and red dots show values for 150 individual cells. The difference between WT and EF1 > coYki 4SA is significant (t-test).

Knockout of coHpo and coWts.

(A) A diagram of the coHpo genomic loci in WT or a putative coHpo -/- knockout line is shown. “KD” indicates kinase domain, “SD” indicates SARAH domain, “GenR” indicates a geneticin resistance cassette, and “NatR” indicates a nourseothricin resistance cassette. Arrows indicate a pair of primers used in PCR reactions. The same primer pair were used in all PCRs shown. Expected PCR product sizes for different alleles are given. A SacII site present only within the allele containing the NatR cassette is shown. (B) PCR products from WT and coHpo -/- cells using primers shown in (A). Because the product sizes for the WT and τιcoHpo::NatR alleles are close in size, we digested PCR products with SacII, which uniquely cuts the τιcoHpo::NatR allele. The coHpo -/- cell line shows the expected products for a homozygous knockout. Sequencing of PCR products confirmed the identity of the WT, τιcoHpo::GenR, and τιcoHpo::NatR alleles. (C) A diagram of the coWts genomic loci in WT or a putative coWts -/- knockout line is shown. “KD” indicates kinase domain, “GenR” indicates a geneticin resistance cassette, and “NatR” indicates a nourseothricin resistance cassette. Arrows indicate a pair of primers used in PCR reactions. The same primer pair were used in all PCRs shown. (D) PCR products from WT and coHpo -/- cells using primers shown in (C). The coWts -/- cell line shows the expected products for a homozygous knockout. Sequencing of PCR products confirmed the identity of the WT, ýcoWts::GenR, and ýcoWts::NatR alleles.