Fos and Jun bend the AP-1 site: effects of probe geometry on the detection of protein-induced DNA bending.

The effect of Fos and Jun binding on the structure of the AP-1 recognition site is controversial. Results from phasing analysis and phase-sensitive detection studies of DNA bending by Fos and Jun have led to opposite conclusions. The differences between these assays, the length of the spacer between two bends and the length of the sequences flanking the bends, are investigated here using intrinsic DNA bend standards. Both an increase in the spacer length as well as a decrease in the length of flanking sequences resulted in a reduction in the phase-dependent variation in electrophoretic mobilities. Probes with a wide separation between the bends and short flanking sequences, such as those used in the phase-sensitive detection studies, displayed no phase-dependent mobility variation. This shape-dependent variation in electrophoretic mobilities was reproduced by complexes formed by truncated Fos and Jun. Results from ligase-catalyzed cyclization experiments have been interpreted to indicate the absence of DNA bending in the Fos-Jun-AP-1 complex. However, truncated Fos and Jun can alter the relative rates of inter- and intramolecular ligation through mechanisms unrelated to DNA bending, confounding the interpretation of cyclization data. The analogous phase- and shape-dependence of the electrophoretic mobilities of the Fos-Jun-AP-1 complex and an intrinsic DNA bend confirm that Fos and Jun bend DNA, which may contribute to their functions in transcription regulation.

Reduction in levels of the cyclin-dependent kinase inhibitor p27kip-1 coupled with transforming growth factor β neutralization induces cell-cycle entry and increases retroviral transduction of primitive human hematopoietic cells

Successful gene therapy depends on stable transduction of hematopoietic stem cells. Target cells must cycle to allow integration of Moloney-based retroviral vectors, yet hematopoietic stem cells are quiescent. Cells can be held in quiescence by intracellular cyclin-dependent kinase inhibitors. The cyclin-dependent kinase inhibitor p15INK4B blocks association of cyclin-dependent kinase (CDK)4/cyclin D and p27kip-1 blocks activity of CDK2/cyclin A and CDK2/cyclin E, complexes that are mandatory for cell-cycle progression. Antibody neutralization of β transforming growth factor (TGFβ) in serum-free medium decreased levels of p15INK4B and increased colony formation and retroviral-mediated transduction of primary human CD34+ cells. Although TGFβ neutralization increased colony formation from more primitive, noncycling hematopoietic progenitors, no increase in M-phase-dependent, retroviral-mediated transduction was observed. Transduction of the primitive cells was augmented by culture in the presence of antisense oligonucleotides to p27kip-1 coupled with TGFβ-neutralizing antibodies. The transduced cells engrafted immune-deficient mice with no alteration in human hematopoietic lineage development. We conclude that neutralization of TGFβ, plus reduction in levels of the cyclin-dependent kinase inhibitor p27, allows transduction of primitive and quiescent hematopoietic progenitor populations.

Phase variation of the lpf operon is a mechanism to evade cross-immunity between Salmonella serotypes

Conventional wisdom holds that phase variation is a mechanism for immune evasion. However, despite fimbrial phase variation, mice previously exposed to Salmonella typhimurium are protected against a subsequent challenge. We evaluated whether lpf phase variation instead may be a mechanism to evade cross-immunity between Salmonella serotypes. Mice were immunized orally with S. typhimurium aroA mutants either that expressed the lpf operon (phase-on variant) or in which the entire lpf operon had been removed by deletion. During a subsequent challenge with virulent Salmonella enteritidis a selection against lpf phase-on variants was observed in mice previously exposed to S. typhimurium long polar fimbriae. Vaccination with S. typhimurium did not confer protection against challenge with S. enteritidis, presumably because lpf phase-off variants were able to evade cross-immunity. We propose that lpf phase variation is a mechanism to evade cross-immunity between Salmonella serotypes, thereby allowing their coexistence in a host population.

A p53-dependent S-phase checkpoint helps to protect cells from DNA damage in response to starvation for pyrimidine nucleotides

Normal mammalian cells arrest primarily in G1 in response to N-(phosphonacetyl)-l-aspartate (PALA), which starves them for pyrimidine nucleotides, and do not generate or tolerate amplification of the CAD gene, which confers resistance to PALA. Loss of p53, accompanied by loss of G1 arrest, permits CAD gene amplification and the consequent formation of PALA-resistant colonies. We have found rat and human cell lines that retain wild-type p53 but have lost the ability to arrest in G1 in response to PALA. However, these cells still fail to give PALA-resistant colonies and are protected from DNA damage through the operation of a second checkpoint that arrests them reversibly within S-phase. This S-phase arrest, unmasked in the absence of the G1 checkpoint, is dependent on p53 and independent of p21/waf1.

Variation in per capita interaction strength: Thresholds due to nonlinear dynamics and nonequilibrium conditions

I measured the strength of interaction between a marine herbivore and its growing resource over a realistic range of absolute and relative abundances. The herbivores (hermit crabs: Pagurus spp.) have slow and/or weak functional and numerical responses to epiphytic diatoms (Isthmia nervosa), which show logistic growth in the absence of consumers. By isolating this interaction in containers in the field, I mimicked many of the physical and biological variables characteristic of the intertidal while controlling the densities of focal species. The per capita effects of consumers on the population dynamics of their resource (i.e., interaction strength) were defined by using the relationship between hermit crab density and proportional change in the resource. When this relationship is fit by a Weibull function, a single parameter distinguishes constant interaction strength from one that varies as a function of density. Constant interaction strength causes the proportion of diatoms to fall linearly or proportionally as hermit crab density increases whereas per capita effects that increase with density cause an accelerating decline. Although many mathematical models of species interactions assume linear dynamics and invariant parameters, at least near equilibrium, the per capita effects of hermit crabs on diatoms varied substantially, apparently crossing a threshold from weak to strong when consumption exceeded resource production. This threshold separates a domain of coexistence from one of local extinction of the resource. Such thresholds may help explain trophic cascades, resource compensation, and context-dependent interaction strengths, while indicating a way to predict trophic effects, despite nonlinearities, as a function of vital rates.

Decoding temporally encoded sensory input by cortical oscillations and thalamic phase comparators

The temporally encoded information obtained by vibrissal touch could be decoded “passively,” involving only input-driven elements, or “actively,” utilizing intrinsically driven oscillators. A previous study suggested that the trigeminal somatosensory system of rats does not obey the bottom-up order of activation predicted by passive decoding. Thus, we have tested whether this system obeys the predictions of active decoding. We have studied cortical single units in the somatosensory cortices of anesthetized rats and guinea pigs and found that about a quarter of them exhibit clear spontaneous oscillations, many of them around whisking frequencies (≈10 Hz). The frequencies of these oscillations could be controlled locally by glutamate. These oscillations could be forced to track the frequency of induced rhythmic whisker movements at a stable, frequency-dependent, phase difference. During these stimulations, the response intensities of multiunits at the thalamic recipient layers of the cortex decreased, and their latencies increased, with increasing input frequency. These observations are consistent with thalamocortical loops implementing phase-locked loops, circuits that are most efficient in decoding temporally encoded information like that obtained by active vibrissal touch. According to this model, and consistent with our results, populations of thalamic “relay” neurons function as phase “comparators” that compare cortical timing expectations with the actual input timing and represent the difference by their population output rate.

Cell cycle-dependent colocalization of BARD1 and BRCA1 proteins in discrete nuclear domains

Germ-line mutations of the BRCA1 gene predispose women to early-onset breast and ovarian cancer by compromising the gene’s presumptive function as a tumor suppressor. Although the biochemical properties of BRCA1 polypeptides are not understood, their expression pattern and subcellular localization suggest a role in cell-cycle regulation. When resting cells are induced to proliferate, the steady-state levels of BRCA1 increase in late G1 and reach a maximum during S phase. Moreover, in S phase cells, BRCA1 polypeptides are hyperphosphorylated and accumulate into discrete subnuclear foci termed “BRCA1 nuclear dots.” BRCA1 associates in vivo with a structurally related protein termed BARD1. Here we show that the steady-state levels of BARD1, unlike those of BRCA1, remain relatively constant during cell cycle progression. However, immunostaining revealed that BARD1 resides within BRCA1 nuclear dots during S phase of the cell cycle, but not during the G1 phase. Nevertheless, BARD1 polypeptides are found exclusively in the nuclear fractions of both G1- and S-phase cells. Therefore, progression to S phase is accompanied by the aggregation of nuclear BARD1 polypeptides into BRCA1 nuclear dots. This cell cycle-dependent colocalization of BARD1 and BRCA1 indicates a role for BARD1 in BRCA1-mediated tumor suppression.

Evolution of foraging behavior in Drosophila by density-dependent selection

One of the rare examples of a single major gene underlying a naturally occurring behavioral polymorphism is the foraging locus of Drosophila melanogaster. Larvae with the rover allele, forR, have significantly longer foraging path lengths on a yeast paste than do those homozygous for the sitter allele, fors. These variants do not differ in general activity in the absence of food. The evolutionary significance of this polymorphism is not as yet understood. Here we examine the effect of high and low animal rearing densities on the larval foraging path-length phenotype and show that density-dependent natural selection produces changes in this trait. In three unrelated base populations the long path (rover) phenotype was selected for under high-density rearing conditions, whereas the short path (sitter) phenotype was selected for under low-density conditions. Genetic crosses suggested that these changes resulted from alterations in the frequency of the fors allele in the low-density-selected lines. Further experiments showed that density-dependent selection during the larval stage rather than the adult stage of development was sufficient to explain these results. Density-dependent mechanisms may be sufficient to maintain variation in rover and sitter behavior in laboratory populations.

Oncogenic stimulation recruits cyclin-dependent kinase in the cell cycle start in rat fibroblast

The rat fibroblast NRK cells are transformed reversibly by a combination of growth factors. When stimulated with serum, NRK cells rely on cyclin-dependent kinase 4 (Cdk4) for their S phase entry. However, when stimulated with serum containing oncogenic growth factors, they come to rely on either Cdk4 or Cdk6, and their S phase entry cannot be blocked unless both Cdk4 and Cdk6 are immunodepleted. Such change of dependence does not occur in the NRK cell mutants defective in an oncogenic signal pathway and, therefore, deficient in anchorage-independent cell cycle start ability, correlating Cdk6 dependence with this remarkable, cancer-associated phenotype. However, both Cdk4 and Cdk6 are activated upon serum stimulation, and neither the amounts of Cdk6, Cdk4, cyclin D1, and cyclin-dependent kinase inhibitors nor the activities or subcellular localization of Cdk6 and Cdk4 are significantly influenced by oncogenic stimulation. Thus, oncogenic stimulation invokes Cdk6 to participate in a critical step of the cell cycle start in a rat fibroblast, but by a mechanism seemingly unrelated to the regulation of the kinase. Given that many hematopoietic cells employ predominantly Cdk6 for the cell cycle start and perform anchorage-independent growth by nature, our results raise the possibility that the oncogenic stimulation-induced anchorage-independent cell cycle start of NRK is elicited by a mechanism similar to the one used for hematopoietic cell proliferation.

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19Ink4d and p27Kip1, in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G2 and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19Ink4d and p27Kip1 cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

The subcellular localization of E2F-4 is cell-cycle dependent

The E2F family of transcription factors plays a crucial role in cell cycle progression. E2F activity is tightly regulated by a number of mechanisms, which include the timely synthesis and degradation of E2F, interaction with retinoblastoma protein family members (“pocket proteins”), association with DP heterodimeric partner proteins, and phosphorylation of the E2F/DP complex. Here we report that another mechanism, subcellular localization, is important for the regulation of E2F activity. Unlike E2F-1, -2, or -3, which are constitutively nuclear, ectopic E2F-4 and -5 were predominantly cytoplasmic. Cotransfection of expression vectors encoding p107, p130, or DP-2, but not DP-1, resulted in the nuclear localization of E2F-4 and -5. Moreover, the transcriptional activity of E2F-4 was markedly enhanced when it was invariably nuclear. Conversely, it was reduced when the protein was excluded from the nucleus, implying that E2F-4 transcription function depends upon its cytological location. In keeping with this, the nuclear/cytoplasmic ratios of endogenous E2F-4 changed as cells exited G0, with high ratios in G0 and early G1 and a progressive increase in cytoplasmic E2F-4 as cells approached S phase. Thus, the subcellular location of E2F-4 is regulated in a cell cycle-dependent manner, providing another potential mechanism for its functional regulation.

Fluid-Phase Markers in the Basolateral Endocytic Pathway Accumulate in Response to the Actin Assembly-promoting Drug Jasplakinolide

To investigate the role of filamentous actin in the endocytic pathway, we used the cell-permeant drug Jasplakinolide (JAS) to polymerize actin in intact polarized Madin–Darby canine kidney (MDCK) cells. The uptake and accumulation of the fluid-phase markers fluorescein isothiocyanate (FITC)-dextran and horseradish peroxidase (HRP) were followed in JAS-treated or untreated cells with confocal fluorescence microscopy, biochemical assays, and electron microscopy. Pretreatment with JAS increased the uptake and accumulation of fluid-phase markers in MDCK cells. JAS increased endocytosis in a polarized manner, with a marked effect on fluid-phase uptake from the basolateral surface but not from the apical surface of polarized MDCK cells. The early uptake of FITC-dextran and HRP was increased more than twofold in JAS-treated cells. At later times, FITC-dextran and HRP accumulated in clustered endosomes in the basal and middle regions of JAS-treated cells. The large accumulated endosomes were similar to late endosomes but they were not colabeled for other late endosome markers, such as rab7 or mannose-6-phosphate receptor. JAS altered transport in the endocytic pathway at a later stage than the microtubule-dependent step affected by nocodazole. JAS also had a notable effect on cell morphology, inducing membrane bunching at the apical pole of MDCK cells. Although other studies have implicated actin in endocytosis at the apical cell surface, our results provide novel evidence that filamentous actin is also involved in the endocytosis of fluid-phase markers from the basolateral membrane of polarized cells.

Calcium-dependent Clustering of Inositol 1,4,5-Trisphosphate Receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcεR1) leads to activation of phospholipase C γ isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5–10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 μM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4–2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36M3R cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4–2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.

Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs

Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity in Xenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles in Xenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.

Characterization of Saccharomyces cerevisiae dna2 Mutants Suggests a Role for the Helicase Late in S Phase

The TOR proteins, originally identified as targets of the immunosuppressant rapamycin, contain an ATM-like “lipid kinase” domain and are required for early G1 progression in eukaryotes. Using a screen to identify Saccharomyces cerevisiae mutants requiring overexpression of Tor1p for viability, we have isolated mutations in a gene we call ROT1 (requires overexpression of Tor1p). This gene is identical to DNA2, encoding a helicase required for DNA replication. As with its role in cell cycle progression, both the N-terminal and C-terminal regions, as well as the kinase domain of Tor1p, are required for rescue of dna2 mutants. Dna2 mutants are also rescued by Tor2p and show synthetic lethality with tor1 deletion mutants under specific conditions. Temperature-sensitive (Ts) dna2 mutants arrest irreversibly at G2/M in a RAD9- and MEC1-dependent manner, suggesting that Dna2p has a role in S phase. Frequencies of mitotic recombination and chromosome loss are elevated in dna2 mutants, also supporting a role for the protein in DNA synthesis. Temperature-shift experiments indicate that Dna2p functions during late S phase, although dna2 mutants are not deficient in bulk DNA synthesis. These data suggest that Dna2p is not required for replication fork progression but may be needed for a later event such as Okazaki fragment maturation.