PMC

Nonmotile cilia in homozygous ACT1Δ homokaryons (A and B) and in a division-arrested somatic ACT1Δ transformant (C) immunostained with antitubulin antibodies. A and P indicate the anterior and posterior ends of cells, respectively. Bar, 10 μm. (See also film B in the supplemental material).

Electron micrographs of sections through cilia having abnormal outer doublet microtubules in somatic ACT1Δ transformants (A to C) and in a homozygous ACT1Δ homokaryon (D). (A) A moderately abnormal cilium with abnormal detachment of B tubules from the A tubules (arrowheads). (B) A more severely abnormal cilium including another example of a detached B tubule (arrowhead). (C) A moderately abnormal cilium with a detached A tubule (arrowhead). (D) A moderately abnormal cilium with a detached B tubule (arrowhead) and an extra incomplete microtubule (arrow). Magnification, 120,000×.

Immunolocalization of ciliary actin. Three frames from the same confocal image of isolated axonemes from homozygous ACT1Δ homokaryons rescued with an MTT-ACT1-3×HA construct and stained with antitubulin (A, fluorescein isothiocyanate) and with anti-HA (C, Alexa Fluor 568). The central panel (B) is an overlay of A and C with a slight displacement to allow comparison of the structures labeled by the two fluorochromes. The arrows in A and B point to a single ciliary axoneme labeled by antitubulin, whereas the arrowheads in B and C indicate the same axoneme labeled by anti-HA. Bar, 10 μm.

Differential interference contrast images of uptake of ink particles in fixed CU428 cells (A and B) and in homozygous ACT1Δ homokaryons (C and D). Arrows indicate ink particles in the region of the oral opening (cytostome), most likely in transit (A) or trapped (C). Cilia fixed in various phases of the normal beat cycle are also faintly visible in A and B, whereas outstretched nonmotile cilia are more easily seen in C and D. Bar, 10 μm. (See also films A and B in the supplemental material).

Cumulative number of cells produced in clones of homozygous ACT1Δ homokaryons cultivated in microdrops in SPP (A) or PPYGFe (B) medium for 15 days, and in subclones cultivated in microdrops of PPYGFe medium for 14 days (C). Each symbol represents a clone or subclone (circles, SPP; squares, PPYGFe), and the position of the symbol on the abscissa indicates the number of cells counted in that clone on day 15 (or 14 for the subclones), or on days 10 to 14 in the few cases when there were late declines in cell number or losses for other reasons. Some but not all of the clones and subclones with 1 to 5 cells became moribund at various times before days 14 to 15, though few cells actually lysed. The three clones used as founders of the subclones are labeled a, b, and c in the histogram (B); they each have their own distinctive symbol, which is used to identify the source of each of the subclones in histogram (C).

Living, nearly immobile cells from cultures of somatic ACT1Δ transformants photographed on the bottom of the microtiter culture wells. (A) A small region at the bottom of a well, showing five cells in various (numbered) stages of furrow regression following arrest at the final stage of cleavage, as well as four nondividing cells. The cell numbered 5 has undergone an anteriorward telescoping of the posterior daughter cell. (B to F) Different cells photographed in successive stages of cleavage and reintegration. (B) Early fission. (C) Completion of furrowing. (D) Re-elongation of presumptive daughter cells that have failed to separate. The posterior daughter is shown pivoted relative to the anterior daughter, probably in an attempt to separate from it. (E) A dividing cell beginning to reintegrate; the connection between the presumptive daughter cells is broadening. (F) A more fully integrated divider, with a broad cytoplasmic union and separated macronuclei (arrowheads). (G to I) Examples of alternative developmental consequences of the permanent reintegration of presumptive daughter cells. (G) An attempted second division of one of the two daughter cells. (H) Displacement of the axes of the two daughter cells. (I) Probable twisting of the two presumptive daughter cells into a heteropolar configuration. All of the photographs are printed at the same magnification. Bar, 50 μm.

Disruption of the ACT1 gene. (A) A diagram showing preparation of the ACT1 disruption construct and the targeting strategy. The neo3 disruption cassette was inserted into the unique SfuI site in the ACT1 coding region of pAct-35-TOPO, thereby creating the pAct-35-TOPO-neo3 targeting construct. The BstXI sites were used to free that construct prior to transformation. The targeting construct replaced the endogenous ACT1 gene by homologous recombination, generating a “knockout allele” that contained a 3,070-bp insertion of the neo3 cassette. (B) Genotypic analysis of the wild-type cells and homozygous ACT1Δ homokaryons. Total genomic DNA isolated from CU428 wild-type cells and ACT1Δ cells was analyzed by PCR using ACT1-specific primers (arrowheads) whose locations are indicated in A. A 1.2-kb band was amplified from the wild-type (WT) cells (center lane). In the homozygous ACT1Δ “knockout” (KO) homokaryons, the 1.2-kb band was absent, and a 4.2-kb major band containing the neo3 insert was obtained (right lane). The left lane shows the molecular size markers. (C) RT-PCR analysis to assay ACT1 expression. Total RNA extracted from CU428 and ACT1Δ cells was analyzed by RT-PCR using the same ACT1-specific primers described in B. A 1.2-kb ACT1 cDNA band (arrowhead) was amplified from the wild-type (WT) cells but not from the ACT1 knockout (KO) cells. For the control, primers specific to ribosomal protein gene RPL21 were included in the same reaction. The 0.36-kb RPL21 cDNA band (asterisk), but not the 0.84-kb band that would be expected from the amplification of contaminating genomic DNA, was obtained from both wild-type and knockout cells. Illustrations were prepared for publication on a Macintosh G4 computer using Photoshop CS software. All adjustments were applied solely to improve the clarity of the images.

Photographs of clones of ACT1ΔC3ns heterokaryons (A) and homozygous ACT1Δ homokaryons (B, C, and D) cultivated in microdrops containing SPP (A, C, and D) or PPYGFe (B) medium. (A) A clone of phenotypically normal (+) heterokaryons, photographed 24 h after clone initiation with a single cell. Approximately 100 swimming cells are uniformly dispersed and therefore mostly out of focus. At least eight cells in various stages of division are in or near the focal plane at the bottom of the drop. (B) A portion of a microdrop containing about one-half of the cells of the largest ACT1Δ clone (a in Fig. ) photographed 14 days after clone initiation. These cells are nearly stationary in a single focal plane just above the plastic surface. Some cells are in various stages of division or integration. Other nondividing cells have single macronuclei and appear nearly normal. (C) An average ACT1Δ clone, photographed 11 days after initiation. All of the 27 nearly stationary cells are in focus just above the plastic substratum. One cell is in late division (arrowhead), another abnormal cell may be budding off a small daughter cell (long thin arrow), and there is one large monster (short thick arrow). Most cells have the irregular rotund shape characteristic of cells that have reintegrated after an aborted cytokinesis, and a few other cells are smaller and more spherical than normal. (D) An ACT1Δ clone, photographed 11 days after clone initiation. All of the cells in the clone are visible in the portion of the drop shown here. A large monster with many cellular subunits is in the process of attempting to bud off a smaller cell (arrow). One small daughter cell had successfully separated from the monster on the eighth day after isolation. A third small cell appeared on the eleventh day. One of these two small cells is attempting to divide again (arrowhead). Bar, 100 μm.

Images of somatic ACT1Δ transformants, immunostained with anticentrin monoclonal antibody 20H5 (A to F and H to I) or polyclonal antitubulin antibody (G). “Left” and “right” refer to the cell's left and right, which is opposite to the viewer's left and right, except in E. (A) A predividing cell with compound ciliary structures of the anterior oral apparatus (OA) and the posterior oral primordium (OP) located along the same longitudinal meridian. Ciliary rows (CR) with basal bodies (BB) are equatorially subdivided to make up a fission zone (FZ), the site of the future division furrow. (B) An optical cross section of a divider in an early stage of reintegration. The division furrow (DF), oral apparatuses (OA1 and OA2), and divided macronuclei (Mac1 and Mac2) are visible. (C) A more fully reintegrated ex-divider (DF, regressed division furrow) with a fully formed oral crescent (OC) of the posterior oral apparatus (OA2) and lateral displacement of OA2 relative to its anterior counterpart (OA1). The presumptive anterior daughter cell possesses a normal anterior crown of basal body couplets (AC1), whereas the anterior end of the posterior daughter cell has only a few brightly stained developing anterior basal body couplets (dAC2). (D) One surface of a partially reintegrated former divider. One ciliary row of the posterior presumptive daughter cell (marked with an arrowhead at its posterior end) appears to be continuous with a ciliary row of the anterior daughter cell. (E) The opposite surface of the same cell as in D. About eight ciliary rows of the posterior daughter cell have formed a partial anterior crown of basal body couplets (AC2). (F) A more completely reintegrated former divider with three ciliary rows (marked a, b, and c) secondarily rejoined across the former fission zone. Other ciliary rows (arrow) have become disrupted and fragmented near the fission zone. (G) Antitubulin staining of a reintegrated cell. OA2 is on the viewer's left side of the cell, and OA1 is on the opposite cell surface and therefore is not visible in this section. Ciliary basal bodies (BB) are indistinctly visible, with prominent longitudinal microtubule bands (LM) immediately to their right, and transverse microtubule bands (TM) extending to their left. Cilia (C), some detached from their cellular moorings, are brightly stained. The LMs on two ciliary rows (marked c and d) appear to be continuous across the former fission zone, and those on a third (marked e) have a small equatorial gap (arrow). Two other ciliary rows, marked a and b, are laterally offset at the fission zone. (H) A failed divider that has attempted to divide again. The less distorted posterior fission zone (arrow) is likely to be the more recent one. (I) An irregular monster representing the end state of successive aborted divisions. An oral apparatus (arrow) has normal membranelles, undulating membrane, and an oral crescent. Bar, 10 μm.