Dynamic formation of ER–PM junctions presents a lipid phosphatase to regulate phosphoinositides

Endoplasmic reticulum–plasma membrane (ER–PM) contact sites play an integral role in cellular processes such as excitation–contraction coupling and store-operated calcium entry (SOCE). Another ER–PM assembly is one tethered by the extended synaptotagmins (E-Syt). We have discovered that at steady state, E-Syt2 positions the ER and Sac1, an integral ER membrane lipid phosphatase, in discrete ER–PM junctions. Here, Sac1 participates in phosphoinositide homeostasis by limiting PM phosphatidylinositol 4-phosphate (PI(4)P), the precursor of PI(4,5)P₂. Activation of G protein–coupled receptors that deplete PM PI(4,5)P₂ disrupts E-Syt2–mediated ER–PM junctions, reducing Sac1’s access to the PM and permitting PM PI(4)P and PI(4,5)P₂ to recover. Conversely, depletion of ER luminal calcium and subsequent activation of SOCE increases the amount of Sac1 in contact with the PM, depleting PM PI(4)P. Thus, the dynamic presence of Sac1 at ER–PM contact sites allows it to act as a cellular sensor and controller of PM phosphoinositides, thereby influencing many PM processes.     

The use of transposons to introduce well-defined deletions in plasmids: Possibilities for in vivo cloning

A method for obtaining well-defined deletions in an octopine Ti plasmid was developed. It was based on the assumption that deletions would occur between two directly repeated transposons, when both are temporarily present in one plasmid molecule. To obtain such a situation, recombination has been forced between Agrobacterium tumefaciens Ti plasmids, each carrying the transposon Tn1831 at a different position. In a number of cases, most probably when the transposons are directly repeated, deletion formation indeed occurred and at high frequency. Mutants were isolated carrying Ti plasmids with one copy of Tn1831, and the region of DNA, between the positions of the transposons in the original plasmid, deleted. Moreover, in the case that the segment of DNA, enclosed by the two transposons, harbors the requirements for autonomous replication of an R plasmid, it is shown that in vivo cloning of such a segment of the Ti plasmid on the R plasmid can be accomplished.     

A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons.

Muscarinic and alpha-adrenergic suppression of current through Ca2+ channels was studied in adult rat superior cervical ganglion neurons using whole-cell and cell-attached configurations of the patch-clamp technique. Oxotremorine methiodide suppressed ICa by both a rapid (much less than 1 s) and a slow (greater than 4 s) process, whereas norepinephrine suppressed ICa only by a rapid process. The slow muscarinic suppression could be prevented by adding 20 mM BAPTA, a Ca2+ chelator, to the recording pipette, whereas the adrenergic suppression was not affected. Muscarinic, but not alpha-adrenergic, receptors can couple to Ca2+ channels by a second messenger capable of diffusing into an on-cell patch. This signal seems not to be carried by intracellular Ca2+, cGMP, cAMP, or protein kinase C.     

Agonists that suppress M-current elicit phosphoinositide turnover and Ca2+ transients, but these events do not explain M-current suppression

The hypothesis that acetylcholine, substance P, and LHRH suppress M-current by activating phospholipase C was tested. Each agonist caused turnover of phosphoinositide, as measured by release of inositol phosphates, and a modest transient rise in intracellular free Ca2+ ([ Ca2+]i), as determined with fura-2. Active phorbol esters depressed M-current only 50% and did not prevent further suppression by LHRH. M-current, its control by agonists, and its depression by phorbol esters were not affected by adding inositol trisphosphate or Ca2+ buffers with high or low Ca2+ to the whole-cell, voltage-clamp pipette. We conclude that phospholipase C activation does occur but does not mediate the suppression of M-current by agonists. Caffeine produced large [Ca2+]i transients and acted as an agonist to suppress M-current.     

The influence of friction and interference on the seating of a hemispherical press-fit cup: a finite element investigation.

The formation of gaps in the polar region of acetabular cups is seen as a drawback of press-fit fixation of non-cemented acetabular cups. Recent findings indicate a link between long-term polar gaps and the gaps present directly after implantation. In this study the process of press-fitting is simulated with a linear-elastic two-dimensional axisymmetric finite-element model. The aim of this paper is to investigate the possible importance of friction and interference on the formation of these gaps. A range of cup-bone friction coefficients (mu = 0.1-0.5) is assigned to the cup-bone interface in order to represent the unknown amount of friction occurring during press-fitting. The cup is modeled with a radius of 27 mm, whereas the radius of the cavity is varied between 26.50 and 26.75 mm, thus, creating 0.50 and 0.25 mm radial interference fits. The difference in cavity radius represents the discrepancy between the radius of the last-reamer-used and radius of the cavity it creates. The subchondral plate is considered as being completely removed during reaming. The effects of impact blows via the surgeon's mallet during surgery are modeled as a series of four load pulses, in which peak force is gradually increased from 0.5 to 4.0 kN. The effects of load removal as well as those of load application are investigated. On load application, the cup penetrates into the cavity, and on load removal, the cup rebounds. Depending on the friction, interference and load applied, the position of the cup after the load pulse is somewhere between its position at peak force and its position at the beginning of the pulse. Although the simplifications and conditions involved in the creation of the model necessitate caution when interpreting the results for all clinical cases, it is found that the seating of hemispherical cups in trabecular bone could be more satisfactory for intermediate values of friction (mu = 0.2-0.3) and smaller interference fits (0.25 mm).     

Diversity of phosphoinositide signaling

Phosphoinositide phospholipids are key membrane signals that activate intrinsic membrane proteins and are loose anchor points for peripheral membrane proteins. They are also precursors for numerous second messengers. This essay reviews historical development, current opinions, and future questions about phosphoinositide signaling.