C.Brakebusch and W.Bergmeier contributed equally to this workPlatelet adhesion on and activation by components of the extracellular matrix are crucial to arrest posttraumatic bleeding, but can also harm tissue by occluding diseased vessels. Integrin a2b1 is thought to be essential for platelet adhesion to subendothelial collagens, facilitating subsequent interactions with the activating platelet collagen receptor, glycoprotein VI (GPVI). Here we show that Cre/loxP-mediated loss of b1 integrin on platelets has no signi®cant effect on the bleeding time in mice. Aggregation of b1-null platelets to native ®brillar collagen is delayed, but not reduced, whereas aggregation to enzymatically digested soluble collagen is abolished. Furthermore, b1-null platelets adhere to ®brillar, but not soluble collagen under static as well as low (150 s ±1 ) and high (1000 s ±1 ) shear ow conditions, probably through binding of aIIbb3 to von Willebrand factor. On the other hand, we show that platelets lacking GPVI can not activate integrins and consequently fail to adhere to and aggregate on ®brillar as well as soluble collagen. These data show that GPVI plays the central role in platelet±collagen interactions by activating different adhesive receptors, including a2b1 integrin, which strengthens adhesion without being essential. Keywords: collagen/Cre/loxP/GPVI/a2b1 integrin/ platelets IntroductionDamage to the integrity of the vessel wall results in exposure of the subendothelial extracellular matrix (ECM), which triggers adhesion and aggregation of platelets (Weiss, 1975). The consequence of this process is the formation of a thrombus, which prevents blood loss at sites of injury or leads to occlusion and irreversible tissue damage or infarction in diseased vessels. Integrins play a central role in adhesion and aggregation of platelets (Phillips et al., 1991;Shattil et al., 1998). Integrins are heterodimeric transmembrane receptors composed of an a and a b subunit. Resting platelets express their integrins in a low af®nity state. After activation, mediated by other platelet receptors, integrins shift to a high af®nity state and bind their ligands ef®ciently (Phillips et al., 1991;Shattil et al., 1998).The ECM of the vessel walls is rich in collagens, which play essential roles in thrombus formation by providing a substrate for platelet adhesion and by activating platelets (Baumgartner, 1977). Besides GPIb-V-IX and aIIbb3 integrin, which interact indirectly with collagen via von Willebrand factor (vWF) (Savage et al., 1998), a large number of collagen receptors have been identi®ed on platelets, including most importantly a2b1 integrin (Santoro, 1986) and glycoprotein VI (GPVI) (Moroi et al., 1989). A current model of platelet adhesion to collagen suggests that the GPIb±vWF interaction mediates initial tethering of platelets at high shear, followed by a2b1 integrin-mediated ®rm adhesion, which halts platelet translocation and allows collagen interactions with GPVI, ®nally resulting in platelet activation and thrombus growth (Sixma et...

Stem cell persistence into adulthood requires self-renewal from early developmental stages. In the developing mouse brain, only apical progenitors located at the ventricle are self-renewing, whereas basal progenitors gradually deplete. However, nothing is known about the mechanisms regulating the fundamental difference between these progenitors. Here we show that the conditional deletion of the small Rho-GTPase cdc42 at different stages of neurogenesis in mouse telencephalon results in an immediate increase in basal mitoses. Whereas cdc42-deficient progenitors have normal cell cycle length, orientation of cell division and basement membrane contact, the apical location of the Par complex and adherens junctions are gradually lost, leading to an increasing failure of apically directed interkinetic nuclear migration. These cells then undergo mitoses at basal positions and acquire the fate of basal progenitors. Thus, cdc42 has a crucial role at the apical pole of progenitors, thereby regulating the position of mitoses and cell fate.

SummaryThe receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding spedfically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell-associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on prolonged treatment with this cytokine are augmented, reflecting an attenuation by the sTNF-Rs of spontaneous TNF activity decay. Evidence that this stabilization of TNF activity by the sTNF-Rs foUows from stabilization of TNF structure within the complexes that TNF forms with the sTNF-Rs is presented here, suggesting that the sTNF-Rs can affect TNF activity not only by interfering with its binding to cells but also by stabilizing its structure and preserving its activity, thus augmenting some of its effects.T NF, a polypeptide cytokine, produced primarily by mononuclear phagocytes, plays a key role in the initiation of the inflammatory response. It has a variety of effects, of different, even contrasting nature (1, 2). Evidence that some of these effects can be detrimental to the host have attracted attention to the mechanisms that regulate TNF function. The intracellular signals for the response to TNF are provided by cell surface receptors, of two distinct molecular species, to which TNF binds at high affinity (3, 4). Both receptors for TNF exist also in soluble forms (5-8), apparently derived by proteolytic cleavage from the cell surface forms (9). These soluble TNF receptors (sTNF-Rs) 1 can compete for TNF with the cell surface receptors and thus block its activity (5-8). It was therefore suggested that they function as physiological attenuators of the activity of TNF, safeguarding against its potentially harmful effects. We report here that the sTNF-Rs affect TNF function also by stabilizing its activity, most likely by preventing dissociation of the homotrimeric TNF molecules (10-13) to inactive monomers. Materials and Methods TNF and Its Soluble Receptors.Recombinant human TNF-c~ (TNF; 6 x 107 U/mg of protein), produced by Genentech Co.1 Abbreviations used in this paper: B-CLL, B-chronic lymphocytic leukemia; sTNF-R, soluble tumor necrosis factor receptor.(San Francisco, CA), was kindly provided by Dr. G. Adolf, of the Boehringer Institute (Vienna, Austria). Radiolabeling of the TNF with mI was performed by the chloramine-T method, as previously described (14). Native TNF was produced by stimulation of human peripheral mononuclear phagocytes with bacterial lipopolysaccharide for 6 h, as described elsewhere (15), and used without further purification. The soluble forms of the type I (p55) and type II (p75) TNF receptors (sTNF-RI and sTNF-RII, previously called TBPI and TBPII [81) were isolated from normal urine by ligand (TNF) affinity purification followed by reversed-phase HPLC, as described before (8).Cells. ...

beta 1 integrins are ubiquitously expressed receptors that mediate cell-cell and cell-extracellular matrix interactions. To analyze the function of beta1 integrin in skin we generated mice with a keratinocyte-restricted deletion of the beta 1 integrin gene using the cre-loxP system. Mutant mice developed severe hair loss due to a reduced proliferation of hair matrix cells and severe hair follicle abnormalities. Eventually, the malformed hair follicles were removed by infiltrating macrophages. The epidermis of the back skin became hyperthickened, the basal keratinocytes showed reduced expression of alpha 6 beta 4 integrin, and the number of hemidesmosomes decreased. Basement membrane components were atypically deposited and, at least in the case of laminin-5, improperly processed, leading to disruption of the basement membrane and blister formation at the dermal-epidermal junction. In contrast, the integrity of the basement membrane surrounding the beta 1-deficient hair follicle was not affected. Finally, the dermis became fibrotic. These results demonstrate an important role of beta 1 integrins in hair follicle morphogenesis, in the processing of basement membrane components, in the maintenance of some, but not all basement membranes, in keratinocyte differentiation and proliferation, and in the formation and/or maintenance of hemidesmosomes.

Integrin receptors connect the extracellular matrix to the actin cytoskeleton. This interaction can be viewed as a cyclical liaison, which develops again and again at new adhesion sites only to cease at sites of de-adhesion. Recent work has demonstrated that multidomain proteins play crucial roles in the integrin±actin connection by providing a high degree of regulation adjusted to the needs of the cell. In this review we present several examples of this paradigm and with special emphasis on the ILK±PINCH±parvin complex, which amply demonstrates how structural and signalling functions are linked together.