Collagen receptors mediate early events in the attachment of epithelial (MDCK) cells

Summary Madin-Darby canine kidney (MDCK) cells kept in suspension culture for 12–15 hr displayed high-affinity binding sites for¹²⁵I-lathyritic (soluble) collagen (120,000/cell,K _D =30nm) and preferred collagens types I and IV over laminin or fibronectin as substrates during the first hour of attachment. On the other hand, after 4 hr, attachment to all four substrates was equally efficient. Upon challenge with a collagen substrate, the high-affinity sites were rapidly recruited on it (T1/2=6 min). Their occupancy by soluble collagen triggered the exocytosis of a second large population of low-affinity collagen binding sites that included laminin and seems to be involved in a second cell-attachment mechanism. These results are compatible with a twostep model of MDCK cell attachment to the substrate: first, via high-affinity collagen binding sites, and second, via laminin of cellular origin.     

Filamin structure,function and mechanics: are altered filamin-mediated force responses associated with human disease?

The cytoskeleton framework is essential not only for cell structure and stability but also for dynamic processes such as cell migration, division and differentiation. The F-actin cytoskeleton is mechanically stabilised and regulated by various actin-binding proteins, one family of which are the filamins that cross-link F-actin into networks that greatly alter the elastic properties of the cytoskeleton. Filamins also interact with cell membrane-associated extracellular matrix receptors and intracellular signalling proteins providing a potential mechanism for cells to sense their external environment by linking these signalling systems. The stiffness of the external matrix to which cells are attached is an important environmental variable for cellular behaviour. In order for a cell to probe matrix stiffness, a mechanosensing mechanism functioning via alteration of protein structure and/or binding events in response to external tension is required. Current structural, mechanical, biochemical and human disease-associated evidence suggests filamins are good candidates for a role in mechanosensing.     

Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

Summary Lysine-rich proteinoids in aqueous solution catalyze the formation of peptides from free amino acids and ATP. This catalytic activity is not found in acidic proteinoids, even though the latter contain some basic amino acid. The pH optimum for the synthesis is about 11, but is appreciable below 8 and above 13. Temperature data indicate an optimum at 20°C or above, with little increase in rate to 60°C. Pyrophosphate can be used instead of ATP, with lesser yields resulting. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous amino acid.Proofs should be sent to S.W. Fox, Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, FL 33134     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.     

Drosophila ATP6AP2/VhaPRR functions both as a novel planar cell polarity core protein and a regulator of endosomal trafficking

Planar cell polarity (PCP) controls the orientation of cells within tissues and the polarized outgrowth of cellular appendages. So far, six PCP core proteins including the transmembrane proteins Frizzled (Fz), Strabismus (Stbm) and Flamingo (Fmi) have been identified. These proteins form asymmetric PCP domains at apical junctions of epithelial cells. Here, we demonstrate that VhaPRR, an accessory subunit of the proton pump V‐ATPase, directly interacts with the protocadherin Fmi through its extracellular domain. It also shows a striking co‐localization with PCP proteins during all pupal wing stages in Drosophila. This localization depends on intact PCP domains. Reversely, VhaPRR is required for stable PCP domains, identifying it as a novel PCP core protein. VhaPRR performs an additional role in vesicular acidification as well as endolysosomal sorting and degradation. Membrane proteins, such as E‐Cadherin and the Notch receptor, accumulate at the surface and in intracellular vesicles of cells mutant for VhaPRR. This trafficking defect is shared by other V‐ATPase subunits. By contrast, the V‐ATPase does not seem to have a direct role in PCP regulation. Together, our results suggest two roles for VhaPRR, one for PCP and another in endosomal trafficking. This dual function establishes VhaPRR as a key factor in epithelial morphogenesis.     

A killer factor produced by the cellular slime mold Polysphondylium pallidum

A killer strain was discovered in cellular slime molds. The wild isolate CK-8 of Polysphondylium pallidum kills all other strains in Polysphondylium and Dictyostelium, as far as could be determined, except strain CK-8 itself and its complementary mating type strain CK-9. Growth-phase cells of CK-8 excrete a killer factor which is sensitive to heat, above 60°C for 5 min, and trypsin. The apparent molecular mass of the factor was determined at 10 000–12 000.Abbreviations BSS Bonner's salt solution - CM conditioned medium     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund