Fig 1.
SPO73 is necessary for proper prospore membrane size.
(A) Images of characteristic prospore membranes (PSMs) in wild-type, spo73Δ, spo71Δ, and spo71Δspo73Δ cells. Upper panel, PSMs marked with GFP-Spo20(51–91), lower panel, DIC. Scale bar, 2 μm. (B) Average PSM perimeters for genotypes shown in A. Error bars are standard error of the mean. Number of PSMs measured for each genotype: WT [319], spo73Δ [176], spo71Δ [257], and spo71Δspo73Δ [100].
Fig 2.
Spo73 localizes to prospore membranes.
(A) Spo73-Envy and Dtr1-BFP imaged in live cells. Spo73-Envy colocalized with the prospore membrane localized Dtr1-BFP. (B) Spo73-Envy expression throughout sporulation. 0hr indicates transfer from sporulation priming media (YPA) into sporulation media (SPO). Pgk1 protein abundance serves as a loading control. Ndt80 expression coincides with expression of mid-sporulation genes, and protein abundance serves as a monitor for progression through sporulation. Meiotic counts were conducted from the same cultures used for protein isolation; 200 cells from each time point were counted. Nuclei were visualized using HTB2-mCherry. % 2 nuclei represents cells entering meiosis and may include cells that will sporulate as dyads, %>2 nuclei represents cells who have progressed into MII. (D) Spo71-myc coimmunoprecipitates with Spo73-Envy. Sporulated cells from wild-type (LH177), Spo71-myc (LH901), Spo73-Envy (LH938), and Spo71-myc+Spo73-Envy (LH1039) strains were immunoprecipitated with GFP and probed for Spo713 using and anti-GFP antibody and for Spo71 using an anti-myc antibody.
Fig 3.
SPO73 acts in opposition to SPO1, similar to SPO71.
(A) The prospore membrane formation defect in spo1Δ cells is partially suppressed by the spo73Δ allele. Class I indicates failure to form 1 or more PSM, Class II indicates 1 or more PSMs formed. Number of asci scored for each genotype: spo1Δ [47], spo73Δspo1Δ [48], and spo73Δspo71Δspo1Δ [41]. (B) Representative images of the types of refractile structures present in mutants. Images are from cells 24–36 hours post-sporulation induction. % indicates frequency of refractile structures. Number of cells scored for each genotype: wild type [900], spo71Δ [938], spo1Δ [1,134], spo73Δ [1,426], spo1Δspo71Δ [1,031], spo1Δspo73Δ [1,094], and triple deletion (spo73Δspo71Δspo1Δ) [945].
Fig 4.
Phosphatidylserine and Spo14 localization confirm prospore membrane phenotypes.
(A) Localization of phosphatidylserine (PS) in wild-type, spo71Δ, spo73Δ and spo1Δ cells. PS was detected using Lact-C2-GFP-p416 [3]. Both elongated and rounded prospore membrane stages are shown for wild-type cells; elongated prospore membranes are not typically seen in spo71Δ and spo73Δ cells (B) Localization of Spo14 in wild-type, spo71Δ, spo73Δ and spo1Δ cells. Spo14 was detected using GFP-Spo14 [26]. For spo1Δ, Class I represents cells without prospore membranes; Class II cells represents cells that make at least one prospore membrane, like in Fig 3. n = 39.
Fig 5.
Loss of VPS13 reduces prospore membrane size more severely than loss of SPO71 or SPO73.
Top photos: DIC images. Bottom photos: PSM visualized using the Spo20 PSM marker, as in Fig 1. PSM average perimeters in μm +/- SEM are given below the genotypes. The number of PSM measured for each genotype are: wild type (319), spo71Δ (257), spo73Δ (176), vps13Δ (110), spo71Δ vps13Δ (136), spo73Δ vps13Δ (101), triple (vps13Δ spo73Δspo71Δ) (100). Scale bar, 2 μm.