HeLa cells prelabelled with [3H]lysoplatelet-activating factor (lyso-PAF) accumulated phosphatidic acid (PtdOH) when incubated in the presence of either propranolol (an inhibitor of PtdOH phosphohydrolase) or phorbol ester. In the presence of ethanol, phorbol ester but not propranolol stimulated the accumulation of phosphatidylethanol, an index of phospholipase D activity. Incubation of cells with [3H]lyso-PAF led to a rapid accumulation of label in diacylglycerol (DG) followed by a delayed accumulation in PtdOH. It is concluded that propranolol-induced PtdOH accumulation is derived from DG by DG kinase and does not involve phospholipase D.
Budding yeast cells exist in two mating types, a and α, which use peptide pheromones to communicate with each other during mating. Mating depends on the ability of cells to polarize up pheromone gradients, but cells also respond to spatially uniform fields of pheromone by polarizing along a single axis. We used quantitative measurements of the response of a cells to α-factor to produce a predictive model of yeast polarization towards a pheromone gradient. We found that cells make a sharp transition between budding cycles and mating induced polarization and that they detect pheromone gradients accurately only over a narrow range of pheromone concentrations corresponding to this transition. We fit all the parameters of the mathematical model by using quantitative data on spontaneous polarization in uniform pheromone concentration. Once these parameters have been computed, and without any further fit, our model quantitatively predicts the yeast cell response to pheromone gradient providing an important step toward understanding how cells communicate with each other.
This paper is associated with a video winner of a 2020 American Physical Society's Division of Fluid Dynamics (DFD) Milton van Dyke Award for work presented at the DFD Gallery of Fluid Motion. The original video is available online at the Gallery of Fluid Motion, https://doi.org/10.1103/APS.DFD.2020.GFM.V0020.
Incubation of HeLa cells for 24 hr with [3H]choline resulted in extensive labeling of the phosphorylcholine and phosphatidylcholine pools. 12-O-Tetradecanoylphorbol-13-acetate (TPA), other phorbol ester tumor promoters, and mezerein stimulated the release of [3H]choline and [3H]phosphorylcholine from such prelabeled cells. The release was accompanied by decreased radioactivity in the phosphorylcholine pool, raising the possibility that the released materials were derived by leakage from this pool. However, TPA did not induce the release of radioactivity from cells containing a prelabeled nucleotide pool. Similarly, the TPA-stimulated release of radioactivity from prelabeled cells closely paralleled the label present in the phospholipid pool rather than the phosphocholine pool. Consequently, it is suggested that the primary source of the released material is phosphatidylcholine acting as a substrate for a phospholipase C enzyme. TPA also simulated the incorporation of [3H]choline into phospholipids, but a time-course study indicated that phospholipase C activation preceded this event. This was supported by the observation that incorporation of [3H]choline was also stimulated by exogenously added phospholipase C.
Organisms must faithfully segregate their chromosomes during cell division; mistakes in this process can be costly and even fatal to the organism (1, 2). During mitosis, replicated chromosomes attach to the spindle, a dynamic system of microtubules organized around two poles. Chromosomes attach to the spindle via kinetochores, structures that form on centromeres and bind the ends of microtubules. For accurate segregation, kinetochores on sister chromosomes must attach to microtubules from opposite poles; incorrect attachments lead to missegregation (3). In PNAS, Umbreit et al. (4) expand our understanding of how kinetochore–microtubule interactions can be regulated to correct improper attachments. The authors use in vitro studies to demonstrate that a component of the kinetochore, the Ndc80 complex, can directly influence the dynamics of the microtubules it is bound to and how the complex can be regulated to correct errors in chromosome attachment.
When platelets bind certain specific ligands they are induced to secrete the contents of their cytoplasmic granules and to aggregate. Studies of the molecular events accompanying this vital physiological response have led to a greater understanding of cell activation in general since the pathways involved are common to a number of cell types. By contrast most of the information about the cell surface molecules that initiate signal transduction has emerged from work on T lymphocyte activation, a process essential to the initiation of the immune response. We have described an activation antigen on T lymphocytes that is involved in the differentiation of these cells. In the present report it is demonstrated that the antigen is expressed on the platelet membrane with about 1,200 copies/platelet. A monoclonal antibody detecting this antigen stimulates platelet secretion and aggregation with a half-maximal response at approximately 10(-8) M. Characterization of the antigen, termed PTA1, reveals a glycoprotein of Mr 67,000 showing extensive N-linked carbohydrate, much of which appears to be heavily sialated. The amino-terminal sequence of PTA1, EEVLWHTSVPFAEXMSLEXVYPSM, indicates that the protein has not previously been characterized. Preliminary investigation of the mechanism by which PTA1 mediates platelet activation suggests involvement of protein kinase C and the 47-kDa protein of platelets is rapidly phosphorylated upon antibody-mediated activation. During this process PTA1 is also phosphorylated, as it is following platelet activation by the other agonists, collagen, thrombin, and 12-O-tetradecanoylphorbol 13-acetate. These results provide the first example of a cell surface glycoprotein that is directly involved in both platelet and T lymphocyte activation.
Understanding the genetic basis of evolutionary adaptation is limited by our ability to efficiently identify the genomic locations of adaptive mutations. Here we describe a method that can quickly and precisely map the genetic basis of naturally and experimentally evolved complex traits using linkage analysis. A yeast strain that expresses the evolved trait is crossed to a distinct strain background and DNA from a large pool of progeny that express the trait of interest is hybridized to oligonucleotide microarrays that detect thousands of polymorphisms between the two strains. Adaptive mutations are detected by linkage to the polymorphisms from the evolved parent. We successfully tested our method by mapping five known genes to a precision of 0.2–24 kb (0.1–10 cM), and developed computer simulations to test the effect of different factors on mapping precision. We then applied this method to four yeast strains that had independently adapted to a fluctuating glucose–galactose environment. All four strains had acquired one or more missense mutations in GAL80, the repressor of the galactose utilization pathway. When transferred into the ancestral strain, the gal80 mutations conferred the fitness advantage that the evolved strains show in the transition from glucose to galactose. Our results show an example of parallel adaptation caused by mutations in the same gene.
The length of the mitotic spindle varies among different cell types. A simple model for spindle length regulation requires balancing two forces: pulling, due to micro-tubules that attach to the chromosomes at their kinetochores, and pushing, due to interactions between microtubules that emanate from opposite spindle poles. In the budding yeast Saccharomyces cerevisiae, we show that spindle length scales with kinetochore number, increasing when kinetochores are inactivated and shortening on addition of synthetic or natural kinetochores, showing that kinetochore-microtubule interactions generate an inward force to balance forces that elongate the spindle. Electron microscopy shows that manipulating kinetochore number alters the number of spindle microtubules: adding extra kinetochores increases the number of spindle microtubules, suggesting kinetochore-based regulation of microtubule number.
'/%3e%3c/defs%3e%3cpath fill='%23012169' d='M0 0h10080v5040H0z'/%3e%3cpath stroke='%23fff' stroke-width='.6' d='m0 0 6 3m0-3L0 3' clip-path='url(%23b)' transform='scale(840)'/%3e%3cpath stroke='%23e4002b' stroke-width='.4' d='m0 0 6 3m0-3L0 3' clip-path='url(%23c)' transform='scale(840)'/%3e%3cpath stroke='%23fff' stroke-width='840' d='M2520 0v2520M0 1260h5040'/%3e%3cpath stroke='%23e4002b' stroke-width='504' d='M2520 0v2520M0 1260h5040'/%3e%3cg fill='%23fff'%3e%3cuse xlink:href='%23d' x='2520' y='3780'/%3e%3cuse xlink:href='%23a' x='7560' y='4200'/%3e%3cuse xlink:href='%23a' x='6300' y='2205'/%3e%3cuse xlink:href='%23a' x='7560' y='840'/%3e%3cuse xlink:href='%23a' x='8680' y='1869'/%3e%3cuse xlink:href='%23e' x='8064' y='2730'/%3e%3c/g%3e%3c/svg%3e)
