Preweaned heifer management on US dairy operations: Part III. Factors associated with Cryptosporidium and Giardia in preweaned dairy heifer calves.

The objective of this study was to evaluate management practices and environmental factors associated with cryptosporidiosis and giardiasis in preweaned heifer calves on US dairy operations. This study was conducted as part of the calf component of the National Animal Health Monitoring System's Dairy 2014 study. The calf component included 104 dairy operations in 13 states and was an 18-mo longitudinal study focused on dairy heifer calves from birth to weaning. Fecal samples were collected from 2,249 calves: 839 calves in the West region (California, Colorado, and Washington) and 1,410 calves in the East region (Iowa, Michigan, Minnesota, Missouri, New York, Ohio, Pennsylvania, Vermont, Virginia, and Wisconsin). Fecal samples were collected only once from calves during the preweaning period. Samples were collected from calves 3 to 66 d of age, with a mean of 22 d. Overall, Cryptosporidium and Giardia were detected in 43.1 and 30.5% of fecal samples, respectively. Backward elimination logistic model selection was used after univariate screening to determine which management practices and environmental factors significantly affected the presence of Cryptosporidium or Giardia. The final Cryptosporidium model included herd size, days of age at fecal collection, and average temperature-humidity index for the month of fecal collection (fTHI). Cryptosporidium was found on a higher percentage of large operations (≥500 cows) than small operations (30 to 99 cows). Younger calves were more likely to have a fecal sample positive for Cryptosporidium than samples from older calves. Fecal samples from calves during the warmer parts of the year (fTHI >70) were more likely to be positive for Cryptosporidium than samples collected in colder months (fTHI <20). The final Giardia model included herd size, days of age at fecal collection, average fTHI, failure of passive transfer status, and average daily gain (kg/d) during the preweaning period. Giardia was isolated more frequently from calves on small operations than on large operations and from calves that were older compared with younger calves. Giardia was more frequently isolated in warmer months. Samples from calves with failure of passive transfer were more likely to have Giardia than calves with adequate passive transfer (>10 g/L IgG). Average daily gain during the preweaning period was lower in calves from which Giardia was isolated. These results highlight the factors associated with the presence of Cryptosporidium and Giardia in preweaned dairy heifer calves.

Preweaned heifer management on US dairy operations: Part V. Factors associated with morbidity and mortality in preweaned dairy heifer calves.

The objective of this study was to evaluate morbidity and mortality in preweaned dairy heifer calves based on different health, feeding, and management practices, as well as environmental factors. This study was conducted as part of the calf component of the National Animal Health Monitoring System's Dairy 2014 study, which included 104 dairy operations in 13 states. The calf component was an 18-mo longitudinal study focused on dairy heifer calves from birth to weaning; data were collected on 2,545 calves. The percentage morbidity for all calves enrolled in the study was 33.9%. Backward elimination model selection was used after univariate screening to determine which management practices and environmental factors significantly affected morbidity and mortality. The final morbidity model included birth weight, serum IgG concentration, ventilation type, and average temperature-humidity index (THI) during the preweaning period. After controlling for other independent variables in the model, calves born at a higher birth weight had a lower predicted risk of morbidity than calves with a lower birth weight. An increase in serum IgG concentration was associated with decreased morbidity. Calves housed in positive- or cross-ventilated systems had a 2.2 times higher odds of developing disease compared with calves housed in natural ventilation systems. Average THI during the preweaning period was inversely correlated with morbidity; as THI increased, the predicted morbidity risk decreased. The percent mortality for all calves enrolled in the study was 5.0%. The final mortality model included birth weight, serum IgG concentration, amount of fat/day in the liquid diet, and morbidity. After controlling for other independent variables in the model, calves born at a higher birth weight had a lower risk of mortality. An increase in serum IgG concentration decreased the risk of mortality. The odds of mortality were 3.1 times higher in calves fed ≤0.15 kg of fat/d in the liquid diet compared with calves fed ≥0.22 kg of fat/d. The odds of mortality were 4.7 times higher in calves that experienced any disease throughout the preweaning period than in calves with no disease. In summary, morbidity and mortality were both associated with birth weight and serum IgG concentration. Additionally, morbidity was associated with ventilation type and average monthly THI, and mortality was associated with amount of fat per day in the liquid diet and morbidity.

Preweaned heifer management on US dairy operations: Part I. Descriptive characteristics of preweaned heifer raising practices.

The objective of this study was to describe preweaned dairy heifer calf management practices on dairy operations across the United States that were used to analyze factors associated with colostrum quality and passive transfer, Cryptosporidium and Giardia, morbidity and mortality, and average daily gain. This study included 104 dairy operations in 13 states that participated in the National Animal Health Monitoring System's Dairy 2014 calf component study. This 18-mo longitudinal study focused on dairy heifer calves from birth to weaning, and data were collected on 2,545 heifer calves. Descriptive statistics were generated regarding colostrum feeding, preweaning housing, milk feeding and consumption, growth, morbidity and mortality, and weaning practices. The majority of calves enrolled were Holsteins (89.4%). Over half the calves (63.2%) enrolled in the study received the majority of their colostrum via bottle; however, 22.1% of calves from 51.0% of operations received colostrum via suckling from their dams. For all calves, the mean time to the first colostrum feeding was 2.8 h, and the average amount of colostrum at the first feeding was 2.9 L, with 4.5 L provided in the first 24 h. The mean serum IgG of all calves was 21.7 g/L; however, 76.0% of operations had at least 1 calf with failure of passive transfer of immunity with a serum IgG below 10 g/L. The majority of calves in the study were housed individually (86.6%). Nonetheless, 20.2% of operations housed some calves in groups, representing 13.4% of all calves. Approximately one-half of the calves in the study (52.3%) were dehorned or disbudded during the preweaning period, with only 27.8% of these calves receiving analgesics or anesthetics during the procedure. Whole or waste milk was the liquid diet type fed to 40.1% of calves, and milk replacer was fed to 34.8% of calves. A combination of milk and milk replacer was fed to 25.1% of calves. Calves, on average, were fed 2.6 L per feeding and fed 2.6 times/d, resulting in a total of 5.6 L of liquid diet fed per day. The mean average daily gain for all calves enrolled in the study was 0.7 kg/d. Fecal samples were collected and almost all operations had at least 1 calf positive for Cryptosporidium (94.2%) or Giardia (99.0%), and 84.6% of operations had calves that tested positive for both Cryptosporidium and Giardia. Over one-third of calves (38.1%) had at least one morbidity event during the preweaning period and the mortality rate was 5.0%. The mean age at weaning was 65.7 d. This study provides an update on dairy heifer raising practices in the United States.

Associations between housing and management practices and the prevalence of lameness, hock lesions, and thin cows on US dairy operations.

The objective of this study was to determine the association among different housing and management practices on the prevalence of lameness, hock lesions, and thin cows on US dairy operations. This study was conducted as part of the National Animal Health Monitoring System's Dairy 2014 study, which included dairy operations in 17 states. Size categories were assigned as follows: small (30-99 cows), medium (100-499 cows), and large (≥500 cows). Trained assessors visited 191 dairy operations from March through July 2014 and recorded locomotion and hock scores (on a 3-point scale), and the number of thin cows (body condition score ≤2.25) from a total of 22,622 cows (average 118 cows per farm). The majority of cows (90.4%) were considered to be sound (locomotion score = 1), 6.9% were mild/moderately lame (locomotion score = 2), and 2.7% were severely lame (locomotion score = 3). Similarly, most cows (87.3%) had no hock lesions (hock score = 1), 10.1% had mild lesions (hock score = 2), and 2.6% had severe hock lesions (hock score = 3). A low percentage of cows (4.2%) were thin. Univariate comparisons were performed using PROC LOGLINK, which accounts for study design and weighting. Variables meeting the univariate screening criterion of P < 0.20 were eligible for entry into multivariable models. Statistical significance in the multivariable models was declared at P < 0.05. Large operations had a lower within-herd prevalence of cows with locomotion score ≥2 and locomotion score = 3 compared with small or medium-sized operations. Operations on which cows were kept primarily on pasture had a lower percentage of locomotion score = 3 than those housed in freestall or open/dry lot operations. The use of sand bedding was associated with a lower within-herd prevalence of locomotion score ≥2 than straw/hay or dry/composted manure as the primary bedding material. Sand bedding was also associated with a lower within-herd prevalence of locomotion score = 3 than other bedding types except for rubber mats or mattresses. Operations that housed cows in an open/dry lot had a lower percentage of hock score ≥2 and hock score = 3 than other housing types. Providing sprinklers for heat abatement and having a nutritionist balance rations for cows was associated with a lower percentage of thin cows. Results from this study highlight management practices that may reduce the prevalence of lameness, hock lesions, and thin cows on dairy operations in the United States.

Dairy cow handling facilities and the perception of Beef Quality Assurance on Colorado dairies.

A survey was conducted on Colorado dairies to assess attitudes and practices regarding Dairy Beef Quality Assurance (DBQA). The objectives were to (1) assess the need for a new handling facility that would allow all injections to be administered via DBQA standards; (2) establish if Colorado dairy producers are concerned with DBQA; and (3) assess differences in responses between dairy owners and herdsmen. Of the 95 dairies contacted, 20 (21%) agreed to participate, with a median herd size of 1,178. When asked to rank the following 7 traits--efficiency, animal safety, human safety, ease of animal handling, ease of operation, inject per Beef Quality Assurance (BQA) procedures, and cost--in order of priority when designing a new handling facility, human and animal safety were ranked highest in priority (first or second) by the majority of participants, with ease of animal handling and efficiency ranked next. Interestingly, the administration of injections per BQA standards was ranked sixth or seventh by most participants. Respondents estimated the average annual income from the sale of cull cows to be 4.6% of all dairy income, with 50% receiving at least one carcass discount or condemnation in the past 12 mo. Although almost all of the participating dairy farmers stated that the preferred injection site for medications was the neck region, a significant number admitted to using alternate injection sites. In contrast, no difference was found between responses regarding the preferred and actual location for intravenous injections. Although most participating producers are aware of BQA injection guidelines, they perceive efficiency as more important, which could result in injections being administered in locations not promoted by BQA. Dairy owners and herdsmen disagreed in whether or not workers had been injured in the animal handling area in the last 12 mo. Handling facilities that allow for an efficient and safe way to administer drugs according to BQA guidelines and educational opportunities that highlight the effect of improved DBQA on profitability could prove useful. Dairy producers play a key role in ensuring that dairy beef is safe and high quality, and just as they are committed to producing safe and nutritious milk for their customers, they should be committed to producing the best quality beef.

Using temperature-sensing reticular boluses to aid in the detection of production diseases in dairy cows.

The objective of this study was to investigate associations between increases in reticular temperature (RT) in dairy cows and the diagnosis of metritis, mastitis, lameness, and pneumonia by dairy personnel. A prospective case-control study was conducted on a 2,175-cow dairy operation in Colorado from May 2010 to April 2011. Each cow received an orally administered temperature sensing reticular bolus after parturition and RT measurements were recorded 3 times per day as lactating cows exited the milking parlor. A cow was identified as having an increased RT when a deviation of 0.8°C above baseline (average of readings of previous 10d) was recorded by the TempTrack software (DVM Systems, LLC, Greeley, CO). During the same study period, dairy personnel without access to RT data recorded health events and classified them according to clinical signs observed. A total of 201 health events (cases) were included in the data analysis. Cows with clinical mastitis and pneumonia had significantly higher odds (6.7 and 7.5 times higher, respectively) of having an increased RT of 0.8°C above their baseline within 4d preceding diagnosis when compared with control cows. Specificity and sensitivity for an increase of 0.8°C above baseline RT within 4d of disease diagnosis was 76.85 and 66.97% for mastitis, and 69.23 and 76.92% for pneumonia, respectively. No significant difference in RT was found for cows diagnosed with lameness or metritis. Results of this study suggest that RT monitoring can be a useful tool in the early detection of mastitis and pneumonia in dairy cows.

Structural rearrangements of tubulin and actin during the cell cycle of the yeast Saccharomyces.

The distribution of actin and tubulin during the cell cycle of the budding yeast Saccharomyces was mapped by immunofluorescence using fixed cells from which the walls had been removed by digestion. The intranuclear mitotic spindle was shown clearly by staining with a monoclonal antitubulin; the presence of extensive bundles of cytoplasmic microtubules is reported. In cells containing short spindles still entirely within the mother cells, one of the bundles of cytoplasmic microtubules nearly always extended to (or into) the bud. Two independent reagents (anti-yeast actin and fluorescent phalloidin) revealed an unusual distribution of actin: it was present as a set of cortical dots or patches and also as distinct fibers that were presumably bundles of actin filaments. Double labeling showed that at no stage in the cell cycle do the distributions of actin and tubulin coincide for any significant length, and, in particular, that the mitotic spindle did not stain detectably for actin. However, both microtubule and actin staining patterns change in a characteristic way during the cell cycle. In particular, the actin dots clustered in rings about the bases of very small buds and at the sites on unbudded cells at which bud emergence was apparently imminent. Later in the budding cycle, the actin dots were present largely in the buds and, in many strains, primarily at the tips of these buds. At about the time of cytokinesis the actin dots clustered in the neck region between the separating cells. These aspects of actin distribution suggest that it may have a role in the localized deposition of new cell wall material.

Relationship of actin and tubulin distribution to bud growth in wild-type and morphogenetic-mutant Saccharomyces cerevisiae.

The distribution of actin in wild-type cells and in morphogenetic mutants of the budding yeast Saccharomyces cerevisiae was explored by staining cells with fluorochrome-labeled phallotoxins after fixing and permeabilizing the cells by several methods. The actin appeared to be localized in a set of cortical spots or patches, as well as in a network of cytoplasmic fibers. Bundles of filaments that may possibly correspond to the fibers visualized by fluorescence were observed with the electron microscope. The putative actin spots were concentrated in small and medium-sized buds and at what were apparently the sites of incipient bud formation on unbudded cells, whereas the putative actin fibers were generally oriented along the long axes of the mother-bud pairs. In several morphogenetic mutants that form multiple, abnormally elongated buds, the actin patches were conspicuously clustered at the tips of most buds, and actin fibers were clearly oriented along the long axes of the buds. There was a strong correlation between the occurrence of active growth at particular bud tips and clustering of actin spots at those same tips. Near the end of the cell cycle in wild-type cells, actin appeared to concentrate (as a cluster of spots or a band) in the neck region connecting the mother cell to its bud. Observations made using indirect immunofluorescence with a monoclonal anti-yeast-tubulin antibody on the morphogenetic mutant cdc4 (which forms multiple, abnormally elongated buds while the nuclear cycle is arrested) revealed the surprising occurrence of multiple bundles of cytoplasmic microtubules emanating from the one duplicated spindle-pole body per cell. It seems that most or all of the buds contain one or more of these bundles of microtubules, which often can be seen to extend to the very tips of the buds. These observations are consistent with the hypotheses that actin, tubulin, or both may be involved in the polarization of growth and localization of cell-wall deposition that occurs during the yeast cell cycle.

Functions of microtubules in the Saccharomyces cerevisiae cell cycle.

We used the inhibitor nocodazole in conjunction with immunofluorescence and electron microscopy to investigate microtubule function in the yeast cell cycle. Under appropriate conditions, this drug produced a rapid and essentially complete disassembly of cytoplasmic and intranuclear microtubules, accompanied by a rapid and essentially complete block of cellular and nuclear division. These effects were similar to, but more profound than, the effects of the related drug methyl benzimidazole carbamate (MBC). In the nocodazole-treated cells, the selection of nonrandom budding sites, the formation of chitin rings and rings of 10-nm filaments at those sites, bud emergence, differential bud enlargement, and apical bud growth appeared to proceed normally, and the intracellular distribution of actin was not detectably perturbed. Thus, the cytoplasmic microtubules are apparently not essential for the establishment of cell polarity and the localization of cell-surface growth. In contrast, nocodazole profoundly affected the behavior of the nucleus. Although spindle-pole bodies (SPBs) could duplicate in the absence of microtubules, SPB separation was blocked. Moreover, complete spindles present at the beginning of drug treatment appeared to collapse, drawing the opposed SPBs and associated nuclear envelope close together. Nuclei did not migrate to the mother-bud necks in nocodazole-treated cells, although nuclei that had reached the necks before drug treatment remained there. Moreover, the double SPBs in arrested cells were often not oriented toward the budding sites, in contrast to the situation in normal cells. Thus, microtubules (cytoplasmic, intranuclear, or both) appear to be necessary for the migration and proper orientation of the nucleus, as well as for SPB separation, spindle function, and nuclear division.

CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae.

Budding in the yeast Saccharomyces cerevisiae involves a polarized deposition of new cell surface material that is associated with a highly asymmetric disposition of the actin cytoskeleton. Mutants defective in gene CDC24, which are unable to bud or establish cell polarity, have been of great interest with regard to both the mechanisms of cellular morphogenesis and the mechanisms that coordinate cell-cycle events. To gain further insights into these problems, we sought additional mutants with defects in budding. We report here that temperature-sensitive mutants defective in genes CDC42 and CDC43, like cdc24 mutants, fail to bud but continue growth at restrictive temperature, and thus arrest as large unbudded cells. Nearly all of the arrested cells appear to begin nuclear cycles (as judged by the occurrence of DNA replication and the formation and elongation of mitotic spindles), and many go on to complete nuclear division, supporting the hypothesis that the events associated with budding and those of the nuclear cycle represent two independent pathways within the cell cycle. The arrested mutant cells display delocalized cell-surface deposition associated with a loss of asymmetry of the actin cytoskeleton. CDC42 maps distal to the rDNA on chromosome XII and CDC43 maps near lys5 on chromosome VII.

Purification of profilin from Saccharomyces cerevisiae and analysis of profilin-deficient cells.

We have isolated profilin from yeast (Saccharomyces cerevisiae) and have microsequenced a portion of the protein to confirm its identity; the region microsequenced agrees with the predicted amino acid sequence from a profilin gene recently isolated from S. cerevisiae (Magdolen, V., U. Oechsner, G. Müller, and W. Bandlow. 1988. Mol. Cell. Biol. 8:5108-5115). Yeast profilin resembles profilins from other organisms in molecular mass and in the ability to bind to polyproline, retard the rate of actin polymerization, and inhibit hydrolysis of ATP by monomeric actin. Using strains that carry disruptions or deletions of the profilin gene, we have found that, under appropriate conditions, cells can survive without detectable profilin. Such cells grow slowly, are temperature sensitive, lose the normal ellipsoidal shape of yeast cells, often become multinucleate, and generally grow much larger than wild-type cells. In addition, these cells exhibit delocalized deposition of cell wall chitin and have dramatically altered actin distributions.

Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin.

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.

A septin-based hierarchy of proteins required for localized deposition of chitin in the Saccharomyces cerevisiae cell wall.

Just before bud emergence, a Saccharomyces cerevisiae cell forms a ring of chitin in its cell wall; this ring remains at the base of the bud as the bud grows and ultimately forms part of the bud scar marking the division site on the mother cell. The chitin ring seems to be formed largely or entirely by chitin synthase III, one of the three known chitin synthases in S. cerevisiae. The chitin ring does not form normally in temperature-sensitive mutants defective in any of four septins, a family of proteins that are constituents of the "neck filaments" that lie immediately subjacent to the plasma membrane in the mother-bud neck. In addition, a synthetic-lethal interaction was found between cdc12-5, a temperature-sensitive septin mutation, and a mutant allele of CHS4, which encodes an activator of chitin synthase III. Two-hybrid analysis revealed no direct interaction between the septins and Chs4p but identified a novel gene, BNI4, whose product interacts both with Chs4p and Cdc10p and with one of the septins, Cdc10p; this analysis also revealed an interaction between Chs4p and Chs3p, the catalytic subunit of chitin synthase III. Bni4p has no known homologues; it contains a predicted coiled-coil domain, but no other recognizable motifs. Deletion of BNI4 is not lethal, but causes delocalization of chitin deposition and aberrant cellular morphology. Overexpression of Bni4p also causes delocalization of chitin deposition and produces a cellular morphology similar to that of septin mutants. Immunolocalization experiments show that Bni4p localizes to a ring at the mother-bud neck that lies predominantly on the mother-cell side (corresponding to the predominant site of chitin deposition). This localization depends on the septins but not on Chs4p or Chs3p. A GFP-Chs4p fusion protein also localizes to a ring at the mother-bud neck on the mother-cell side. This localization is dependent on the septins, Bni4p, and Chs3p. Chs3p, whose normal localization is similar to that of Chs4p, does not localize properly in bni4, chs4, or septin mutant strains or in strains that accumulate excess Bni4p. In contrast, localization of the septins is essentially normal in bni4, chs4, and chs3 mutant strains and in strains that accumulate excess Bni4p. Taken together, these results suggest that the normal localization of chitin synthase III activity is achieved by assembly of a complex in which Chs3p is linked to the septins via Chs4p and Bni4p.

A yeast actin-binding protein is encoded by SAC6, a gene found by suppression of an actin mutation.

The protein encoded by SAC6, a gene that can mutate to suppress a temperature-sensitive defect in the yeast actin gene, has been identified as a 67-kilodalton actin-binding protein (ABP 67) that associates with all identifiable actin structures. This finding demonstrates the in vivo functional importance of the actin-ABP 67 interaction previously established in vitro and illustrates the use of suppressor analysis to identify physically interacting proteins.

Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin.

The actin cytoskeleton is a fundamental component of eukaryotic cells, with both structural and motile roles. Actin and many of the actin-binding proteins found in different cell types are highly conserved, showing considerable similarity in both primary structure and biochemical properties. To make detailed comparisons between homologous proteins, it is necessary to know whether the various proteins are functionally, as well as structurally, conserved. Fimbrin is an example of a cytoskeletal component that, as shown by sequence determinations and biochemical characterizations, is conserved between organisms as diverse as Saccharomyces cerevisiae and humans. In this study, we examined whether the human homolog can substitute for the yeast protein in vivo. We report here that two isoforms of human fimbrin, also referred to as T- and L-plastin, can both substitute in vivo for yeast fimbrin, also known as Sac6p, whereas a third isoform, I-fimbrin (or I-plastin), cannot. We demonstrate that the human T- and L-fimbrins, in addition to complementing the temperature-sensitive growth defect of the sac6 null mutant, restore both normal cytoskeletal organization and cell shape to the mutant cells. In addition, we show that human T- and L-fimbrins can complement a sporulation defect caused by the sac6 null mutation. These findings indicate that there is a high degree of functional conservation in the cytoskeleton, even between organisms as diverse as S. cerevisiae and humans.

Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.

Dominant suppressors of yeast actin mutations that are reciprocally suppressed.

A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.

Genetic analysis of the fimbrin-actin binding interaction in Saccharomyces cerevisiae.

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have sequenced 17 sac6 suppressor alleles, and found that they change nine different residues, all of which cluster in three regions of one of the two actin-binding domains of Sac6p. Two of these clusters occur in highly conserved regions (ABS1 and ABS3) that have been strongly implicated in the binding of related proteins to actin. The third cluster changes residues not previously implicated in the interaction with actin. As changes in any of nine different residues can suppress several different act1 alleles, it is likely that the suppressors restore the overall affinity, rather than specific lost interactions, between Sac6p and actin. Using mutagenesis, we have identified two mutations of the second actin-binding domain that can also suppress the act1 mutations of interest. This result suggests the two actin-binding domains of Sac6p interact with the same region of the actin molecule. However, differences in strength of suppression of temperature-sensitivity and sporulation indicate that the two actin-binding domains are distinct, and explain why second-domain mutations were not identified previously.

Allele-specific suppression by formation of new protein-protein interactions in yeast.

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have conducted a biochemical analysis of the interaction between various combinations of wild-type and mutant actin and Sac6 proteins. Previously, we showed that actin mutations that are suppressed by sac6 mutations encode proteins with a reduced affinity for wild-type Sac6p. In the present study, we have found that mutant Sac6 proteins bind more tightly to mutant actin than does wild-type Sac6p, and thus compensate for weakened interactions caused by the mutant actin. Remarkably, we have also found that mutant Sac6 proteins bind more tightly to wild-type actin than does wild-type Sac6p. This result indicates that suppression does not occur through the restoration of the original contact site, but rather through the formation of a novel contact site. This finding argues against suppression occurring through a "lock-and-key" mechanism and suggests a mechanism involving more global increases in affinity between the two proteins. We propose that the most common kind of suppressors involving interacting proteins will likely occur through this less specific mechanism.