The ability to express or deplete proteins in living cells is crucial for the study of biological processes. Viral vectors are often useful to deliver DNA constructs to cells that are difficult to transfect by other methods. Lentiviruses have the additional advantage of being able to integrate into the genomes of non-dividing mammalian cells. However, existing viral expression systems generally require different vector backbones for expression of cDNA, small hairpin RNA (shRNA) or microRNA (miRNA) and provide limited drug selection markers. Furthermore, viral backbones are often recombinogenic in bacteria, complicating the generation and maintenance of desired clones. Here, we describe a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. These vectors are based on the Gateway technology (Invitrogen) whereby the cDNA, shRNA or miRNA of interest is cloned into an Entry vector and then recombined into a Destination vector that carries the chosen viral backbone and drug selection marker. This recombination reaction generates the desired product with >95% efficiency and greatly reduces the frequency of unwanted recombination in bacteria. We generated Destination vectors for the production of both retroviruses and lentiviruses. Further, we characterized each vector for its viral titer production as well as its efficiency in expressing or depleting proteins of interest. We also generated multiple types of vectors for the production of fusion proteins and confirmed expression of each. We demonstrated the utility of these vectors in a variety of functional studies. First, we show that the FKBP12 Destabilization Domain system can be used to either express or deplete the protein of interest in mitotically-arrested cells. Also, we generate primary fibroblasts that can be induced to senesce in the presence or absence of DNA damage. Finally, we determined that both isoforms of the AT-Rich Interacting Domain 4B (ARID4B) protein could induce G1 arrest when overexpressed. As new technologies emerge, the vectors in this collection can be easily modified and adapted without the need for extensive recloning.

The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.

Acetylation of histone H3 on lysine 56 occurs during mitotic and meiotic S phase in fungal species. This acetylation blocks a direct electrostatic interaction between histone H3 and nucleosomal DNA, and the absence of this modification is associated with extreme sensitivity to genotoxic agents. We show here that H3-K56 acetylation is catalyzed when Rtt109, a protein that lacks significant homology to known acetyltransferases, forms an active complex with either of two histone binding proteins, Asf1 or Vps75. Rtt109 binds to both these cofactors, but not to histones alone, forming enzyme complexes with kinetic parameters similar to those of known histone acetyltransferase (HAT) enzymes. Therefore, H3-K56 acetylation is catalyzed by a previously unknown mechanism that requires a complex of two proteins: Rtt109 and a histone chaperone. Additionally, these complexes are functionally distinct, with the Rtt109/Asf1 complex, but not the Rtt109/Vps75 complex, being critical for resistance to genotoxic agents.

No abstract

Chromaffin cells of the adrenal medulla are a primary neuroendocrine output of the sympathetic nervous system. When stimulated, they secrete a host of transmitter molecules, including catecholamines and neuropeptides, through the fusion of dense core secretory granules with the cell surface. At basal firing rates, set by the sympathetic tone, chromaffin cells selectively release catecholamines at a modest rate. Stress-mediated sympathetic activation leads to elevated catecholamine secretion and also evokes neuropeptide release. Catecholamines and neuropeptides are copackaged in the same granules; thus, it is unclear how this activity-dependent differential transmitter release is achieved. In this report, we use electrophysiological, electrochemical, fluorescence, and immunocytochemical approaches to quantify transmitter release under physiological electrical stimulation at the single cell level. We provide data to show that chromaffin cells selectively release catecholamine under basal firing conditions but release both neuropeptides and catecholamines under conditions that match acute stress. We further show that this differential transmitter release is achieved through a regulated activity-dependent dilation of the granule fusion pore. Thus, chromaffin cells may regulate release of different transmitters through a simple size-exclusion mechanism.

Recovery from depletion of the readily releasable pool of vesicles (RRP) in adrenal chromaffin cells was studied at differing basal [Ca2+]i or following protein kinase C (PKC) activation by phorbol esters. Following depletion, the pool size was estimated at varied times from cell capacitance jumps in response to paired depolarizations. The experimentally observed RRP recovery time course and steady-state size could be predicted from the measured [Ca2+]i signal assuming a Michaelis-Menten-type regulation of the vesicle supply by Ca2+. An elevated recruitment activity was observed at increased [Ca2+]i even when protein kinase C was blocked, but maximum effects could be obtained only after stimulation of PKC by phorbol esters or by prolonged elevations in [Ca2+]i. We suggest that, in chromaffin cells, elevated cytosolic Ca2+ modulates exocytotic plasticity via PKC-dependent and -independent pathways.

ATP-dependent chromatin-remodeling complexes (remodelers) modulate gene transcription by regulating the accessibility of highly packaged genomic DNA. However, the molecular mechanisms involved at the nucleosomal level in this process remain controversial. Here, we monitor the real-time activity of single ySWI/SNF or RSC complexes on single, stretched nucleosomal templates under tensions above 1 pN forces. We find that these remodelers can translocate along DNA at rates of approximately 13 bp/s and generate forces up to approximately 12 pN, producing DNA loops of a broad range of sizes (20-1200 bp, average approximately 100 bp) in a nucleosome-dependent manner. This nucleosome-specific activity differs significantly from that on bare DNA observed under low tensions and suggests a nucleosome-remodeling mechanism through intranucleosomal DNA loop formation. Such loop formation may provide a molecular basis for the biological functions of remodelers.

We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.