General aspects of peptide selectivity towards lipid bilayers and cell membranes studied by variation of the structural parameters of amphipathic helical model peptides.

Model compounds of modified hydrophobicity (Eta), hydrophobic moment (mu) and angle subtended by charged residues (Phi) were synthesized to define the general roles of structural motifs of cationic helical peptides for membrane activity and selectivity. The peptide sets were based on a highly hydrophobic, non-selective KLA model peptide with high antimicrobial and hemolytic activity. Variation of the investigated parameters was found to be a suitable method for modifying peptide selectivity towards either neutral or highly negatively charged lipid bilayers. Eta and mu influenced selectivity preferentially via modification of activity on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) bilayers, while the size of the polar/hydrophobic angle affected the activity against 1-palmitoyl-2-oleoylphosphatidyl-DL-glycerol (POPG). The influence of the parameters on the activity determining step was modest in both lipid systems and the activity profiles were the result of the parameters' influence on the second less pronounced permeabilization step. Thus, the activity towards POPC vesicles was determined by the high permeabilizing efficiency, however, changes in the structural parameters preferentially influenced the relatively moderate affinity. In contrast, intensive peptide accumulation via electrostatic interactions was sufficient for the destabilization of highly negatively charged POPG lipid membranes, but changes in the activity profile, as revealed by the modification of Phi, seem to be preferentially caused by variation of the low permeabilizing efficiency. The parameters proved very effective also in modifying antimicrobial and hemolytic activity. However, their influence on cell selectivity was limited. A threshold value of hydrophobicity seems to exist which restricted the activity modifying potential of mu and Phi on both lipid bilayers and cell membranes.     

Mechanochemical coupling in spin-labeled, active, isometric muscle

Observed effects of inorganic phosphate (P(i)) on active isometric muscle may provide the answer to one of the fundamental questions in muscle biophysics: how are the free energies of the chemical species in the myosin-catalyzed ATP hydrolysis (ATPase) reaction coupled to muscle force?. Pflugers Arch. 414:73-81) showed that active, isometric muscle force varies logarithmically with [P(i)]. Here, by simultaneously measuring electron paramagnetic resonance and the force of spin-labeled muscle fibers, we show that, in active, isometric muscle, the fraction of myosin heads in any given biochemical state is independent of both [P(i)] and force. These direct observations of mechanochemical coupling in muscle are immediately described by a muscle equation of state containing muscle force as a state variable. These results challenge the conventional assumption mechanochemical coupling is localized to individual myosin heads in muscle.     

Real-time imaging of the dynamics of secretory granules in growth cones

Secretory granules containing a hybrid protein consisting of the regulated secretory protein tissue plasminogen activator and an enhanced form of green fluorescent protein were tracked at high spatial resolution in growth cones of differentiated PC12 cells. Tracking shows that granules, unlike synaptic vesicles, generally are mobile in growth cones. Quantitative analysis of trajectories generated by granules revealed two dominant modes of motion: diffusive and directed. Diffusive motion was observed primarily in central and peripheral parts of growth cones, where most granules diffused two to four orders of magnitude more slowly than comparably sized spheres in dilute solution. Directed motion was observed primarily in proximal parts of growth cones, where a subset of granules underwent rapid, directed motion at average speeds comparable to those observed for granules in neurites. This high-resolution view of the dynamics of secretory granules in growth cones provides insight into granule organization and release at nerve terminals. In particular, the mobility of granules suggests that granules, unlike synaptic vesicles, are not tethered stably to cytoskeletal structures in nerve terminals. Moreover, the slow diffusive nature of this mobility suggests that secretory responses involving centrally distributed granules in growth cones will occur slowly, on a time scale of minutes or longer.     

A nucleolar auto-antigen is part of a major chromosomal surface component

Several nucleolar antigens are defined by human autoantibodies. These antigens can therefore be used to follow the fate of nucleolar components through mitosis when this major nuclear structure disintegrates and becomes reassembled in G₁-phase. We found that fibrillarin leaves the nucleolus before complete breakdown of this structure and attaches to chromosomes before nuclear envelope breakdown. In mouse, fibrillarin attaches over the chromosomal surface except for the excluded centromeric region. The antigen is transported to the new nucleus via the chromosomes and is last seen on chromosomal surfaces facing the cytoplasm during nuclear envelope reformation. Lamin B reappears on the same chromosomal surfaces before the nucleolar antigen is removed and aggregates for new nucleolar reformation in G₁-phase cells. From our observations, we postulate that the antigen acts in concert with other proteins as a nuclear envelope equivalent by forming a protective sheath around the chromosome, that it excludes larger molecules, and helps to separate the chromosomes, in addition to segregation of the ribonucleoprotein (RNP) back to the nucleus for nucleolar reconstruction. We also suggest that the selective retention of these antigens from certain areas on individual chromosomes together with specific lamin B attachment over these chromosomal surfaces allows for a nonrandom positioning of chromosomes in the nucleus.     

Cytoplasmic modification of the nuclear lamina during pronuclear-like transformation of mouse blastomere nuclei.

During the successive interphases of cleaving mouse embryos the nuclear periphery diminishes its reactivity to anti-lamin A and C antibodies. This developmentally regulated characteristic can be modified by exposure of the blastomere nuclei to metaphase II (M II) oocyte cytoplasm followed by activation. In the current study we define the cytoplasmic conditions necessary for this modification of 8-cell and 16-cell stage nuclei in hybrids obtained by fusion with metaphase II arrested oocytes, oocytes at various time points after parthenogenetic activation, naturally fertilized eggs (zygotes) and interphase 2-cell embryo blastomeres. The intensity of fluorescence obtained with anti-lamins A/C in the blastomere nuclei increases as a result of fusion with freshly activated oocytes or early zygotes (first 3.0-5.5 h in the case of parthenogenetic activation), and not when eggs or 2-cell blastomeres advanced in interphase are used as partners for fusion. This transformation of the A/C lamin pattern is correlated with the ability to promote pronucleus-like growth of blastomere nuclei in hybrids. Blastomere nuclei introduced into M II-arrested oocytes undergo premature chromatin condensation and dissolution of the nuclear lamina. The results are discussed with regard to certain particularities of the first embryonic interphase of the mouse and the potential involvement of nuclear lamins in pronuclear growth.     

EFFECT OF METABOLIC INHIBITORS ON NUCLEAR PORE FORMATION DURING THE HELA S3 CELL CYCLE

The effect of various antimetabolites on nuclear pore formation was studied in synchronized HeLa S₃ cells. The nuclear size was determined by light microscopy and the pore number per unit area of nuclear surface by the freeze-etching technique and electron microscopy. It was found that the inhibition of DNA replication or ribosomal RNA synthesis has no effect on nuclear size increase or pore formation. However, the inhibition of ATP synthesis effectively stops nuclear pore formation. Cycloheximide blocks nuclear pore formation at the same time during G₁ phase of the cell cycle when nuclear size increase is blocked by high concentrations of actinomycin D. This suggests that certain proteins or other factors leading to pore formation and nuclear size increase are transcribed and synthesized at about 3–4 h after mitosis, i.e., about 1–2 h before S phase begins.