Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6mg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

Dexamethasone inhibition of leucocyte adhesion to rat mesenteric postcapillary venules: role of intercellular adhesion molecule 1 and KC

BACKGROUND A previous study showed that the glucocorticoid dexamethasone, at doses of 100 ¿g/kg and above, inhibited leucocyte adhesion to rat mesenteric postcapillary venules activated with interleukin 1ß (IL-1ß), as assessed by videomicroscopy. AIMS To identify whether the adhesion molecule, intercellular adhesion molecule 1 (ICAM-1), or the chemokine KC could be targeted by the steroid to mediate its antiadhesive effect. METHODS Rat mesenteries were treated with IL-1ß (20 ng intraperitoneally) and the extent of leucocyte adhesion measured at two and four hours using intravital microscopy. Rats were treated with dexamethasone, and passively immunised against ICAM-1 or KC. Endogenous expression of these two mediators was validated by immunohistochemistry, ELISA, and the injection of specific radiolabelled antibodies. RESULTS Dexamethasone greatly reduced IL-1ß induced leucocyte adhesion, endothelial expression of ICAM-1 in the postcapillary venule, and release of the mast cell derived chemokine KC. Injection of specific antibodies to the latter mediators was also extremely effective in downregulating (>80%) IL-1ß induced leucocyte adhesion. CONCLUSIONS Induction by IL-1ß of endogenous ICAM-1 and KC contributes to leucocyte adhesion to inflamed mesenteric vessels. Without excluding other possible mediators, these data clearly show that dexamethasone interferes with ICAM-1 expression and KC release from mast cells, resulting in suppression of leucocyte accumulation in the bowel wall, which is a prominent feature of several gastrointestinal pathologies.

Podocalyxin is a novel polysialylated neural adhesion protein with multiple roles in neural development and synapse formation

Neural development and plasticity are regulated by neural adhesion proteins, including the polysialylated form of NCAM (PSA-NCAM). Podocalyxin (PC) is a renal PSA-containing protein that has been reported to function as an anti-adhesin in kidney podocytes. Here we show that PC is widely expressed in neurons during neural development. Neural PC interacts with the ERM protein family, and with NHERF1/2 and RhoA/G. Experiments in vitro and phenotypic analyses of podxl-deficient mice indicate that PC is involved in neurite growth, branching and axonal fasciculation, and that PC loss-of-function reduces the number of synapses in the CNS and in the neuromuscular system. We also show that whereas some of the brain PC functions require PSA, others depend on PC per se. Our results show that PC, the second highly sialylated neural adhesion protein, plays multiple roles in neural development.

Ultrastructure of adhesion and movement of the tetraspores of Gelidium Lamour (Geldiaceae; Rhodophyta)

The outer part of the tetraspora cell wall in Gelidium crinale (Turner) J.V. Lamour. and G. spathulatum (Kutz.) Bornet is morphologically described in relation to the movements and displacement of these spores when they settle on a substratum. We also describe the mechanism of adhesión and the transformations undergone by this mechanism over time. The cell wall shows a network of fibrillar threads embedded in abundant mucilage. The deformations that tetraspores undergo show that the cell wall is relatively elastic.

PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3)

The human PFKFB3 is composed of 19 exons spanning genomic region about 90,6 Kb (GenBank). Alternative splicing variants have been reported. The main variants corresponding to mRNAs of 4453 bp and 4224 bp for the variant 1 u-PFK2 (NM_004566.3) and variant 2 i-PFK2 (NM_001145443.1), respectively...

Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions

Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB¿/¿ mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA¿/¿ MEFs, but not FlnB¿/¿ MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Optimized back-focal-plane interferometry directly measures forces of optically trapped particles

Back-focal-plane interferometry is used to measure displacements of optically trapped samples with very high spatial and temporal resolution. However, the technique is closely related to a method that measures the rate of change in light momentum. It has long been known that displacements of the interference pattern at the back focal plane may be used to track the optical force directly, provided that a considerable fraction of the light is effectively monitored. Nonetheless, the practical application of this idea has been limited to counter-propagating, low-aperture beams where the accurate momentum measurements are possible. Here, we experimentally show that the connection can be extended to single-beam optical traps. In particular, we show that, in a gradient trap, the calibration product κ·β (where κ is the trap stiffness and 1/β is the position sensitivity) corresponds to the factor that converts detector signals into momentum changes; this factor is uniquely determined by three construction features of the detection instrument and does not depend, therefore, on the specific conditions of the experiment. Then, we find that force measurements obtained from back-focal-plane displacements are in practice not restricted to a linear relationship with position and hence they can be extended outside that regime. Finally, and more importantly, we show that these properties are still recognizable even when the system is not fully optimized for light collection. These results should enable a more general use of back-focal-plane interferometry whenever the ultimate goal is the measurement of the forces exerted by an optical trap.

Optimized back-focal-plane interferometry directly measures forces of optically trapped particles

Back-focal-plane interferometry is used to measure displacements of optically trapped samples with very high spatial and temporal resolution. However, the technique is closely related to a method that measures the rate of change in light momentum. It has long been known that displacements of the interference pattern at the back focal plane may be used to track the optical force directly, provided that a considerable fraction of the light is effectively monitored. Nonetheless, the practical application of this idea has been limited to counter-propagating, low-aperture beams where the accurate momentum measurements are possible. Here, we experimentally show that the connection can be extended to single-beam optical traps. In particular, we show that, in a gradient trap, the calibration product κ·β (where κ is the trap stiffness and 1/β is the position sensitivity) corresponds to the factor that converts detector signals into momentum changes; this factor is uniquely determined by three construction features of the detection instrument and does not depend, therefore, on the specific conditions of the experiment. Then, we find that force measurements obtained from back-focal-plane displacements are in practice not restricted to a linear relationship with position and hence they can be extended outside that regime. Finally, and more importantly, we show that these properties are still recognizable even when the system is not fully optimized for light collection. These results should enable a more general use of back-focal-plane interferometry whenever the ultimate goal is the measurement of the forces exerted by an optical trap.

Holographic optical tweezers combined with back-focal-plane displacement detection

A major problem with holographic optical tweezers (HOTs) is their incompatibility with laser-based position detection methods, such as back-focal-plane interferometry (BFPI). The alternatives generally used with HOTs, like high-speed video tracking, do not offer the same spatial and temporal bandwidths. This has limited the use of this technique in precise quantitative experiments. In this paper, we present an optical trap design that combines digital holography and back-focal-plane displacement detection. We show that, with a particularly simple setup, it is possible to generate a set of multiple holographic traps and an additional static non-holographic trap with orthogonal polarizations and that they can be, therefore, easily separated for measuring positions and forces with the high positional and temporal resolutions of laser-based detection. We prove that measurements from both polarizations contain less than 1% crosstalk and that traps in our setup are harmonic within the typical range. We further tested the instrument in a DNA stretching experiment and we discuss an interesting property of this configuration: the small drift of the differential signal between traps.

Glibenclamide enhances neurogenesis and improves long-term functional recovery after transient focal cerebral ischemia

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6mg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3)

The human PFKFB3 is composed of 19 exons spanning genomic region about 90,6 Kb (GenBank). Alternative splicing variants have been reported. The main variants corresponding to mRNAs of 4453 bp and 4224 bp for the variant 1 u-PFK2 (NM_004566.3) and variant 2 i-PFK2 (NM_001145443.1), respectively...