Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.

Labeling of peroxisomes with green fluorescent protein in living P. pastoris cells.

We exploited the light-activated fluorescent properties of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria for studies on the peroxisomal sorting of polypeptides. GFP and GFP-SKL (containing a C-terminal, tripeptide peroxisomal targeting signal, SKL) were expressed from a methanol-inducible, alcohol oxidase (AOX1) promoter in the methylotrophic yeast Pichia pastoris. GFP was cytosolic, whereas the GFP-SKL fusion protein was targeted to peroxisomes, as demonstrated by biochemical fractionation of organelles on Nycodenz gradients. Neither GFP nor GFP-SKL affected the viability of yeast cells but both were fluorescent on excitation with 395-nm UV light. The subcellular locations of GFP and GFP-SKL in living yeast cells were monitored by fluorescence microscopy and their fluorescence was coupled to photo-oxidation of diaminobenzidine (DAB), resulting in the deposition of electron-dense oxidized DAB at intracellular locations of GFP derivatives. This photooxidation procedure permitted facile ultrastructural localization of GFP in cells by electron microscopy, and provided further evidence that GFP produced in P. pastoris is cytosolic, whereas GFP-SKL is peroxisomal. The GFP-SKL fusion protein is therefore a versatile reporter for the peroxisomal compartment, with many applications for studies involving peroxisomal import and biogenesis.

[Devices facilitating the preparation of biological specimens for electron microscopy]. / Prisposobleniia, oblegchaiushchie podgotovku biologicheskikh obraztsov dlia élektronnoi mikroskopii.

The construction of a matrix is described which facilitates the process of placing biological objects into polymer media in order to prepare ultrathin sections without the employment of gelatin, starch and polyethylene capsules that can be used only once. The construction of a reactor for cytochemical assays is presented. The apparatus can be used to locate enzymes within the cell, and to identify microorganisms. A modification of the dialysis technique for microbiological objects is proposed which accelerates and simplifies the process.

[Morphology and growth cycle of Hyphomicrobium with a screw-like prostheca]. / Morfologiia i tsikl razvitiia Hyphomicrobium s vintoobraznoi prostekoi

Hyphomicrobium with a screw-like prostheca was isolated from a mixed culture of soil bacteria. Its morphology and growth cycle were studied by electron microscopy. The adult organisms are 1.6-1.8 mcm long and 0.8 mcm thick. The diameter of the prostheca is 0.2-0.3 mcm and sometimes up to 10-12 mcm. It has a peculiar screw-like structure of the cell wall surface and forms branches at whose ends daughter organisms develop. The bacterium multiplies not only by vegetative growth but also by conjugation.

[Localization of energy generators in methane oxidizing bacteria]. / Lokalizatsiia énergogeneratorov u metanokisliaiushchikh bakterii

Cytochromes a, b and c, and their quantitative distribution in the cells, were studied by means of differential spectra in obligate methane oxidizing bacteria, Methylosinus trichosporium with the serine pathway of methane carbon assimilation and Methylomonas agile with the ribulose phosphate pathway of methane carbon assimilation, and different types of topography of intracytoplasmic membranes. The membranes are involved in processes of coupled respiration which was confirmed by cytochemical reactions employed for studying the terminal step of the electron transport chain and Mg2+-stimulated ATPase.

The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis.

Purple sulfur bacterium Thiocapsa roseopersicina strain BBS requiring vitamin B12 may grow in the dark in media containing no other organic compounds. Under such conditions the cells oxidize sulfide and thiosulfate with the use of O2 and assimilate carbon dioxide. After 10--30s assimilation of NaH14CO3 about 60% of radioactivity is found in phosphorylated compounds characteristic for the reductive pentose phosphate cycle. The possibility of the function of this cycle in the dark in the presence of O2 is confirmed by the capacity of cells grown under such conditions to synthesize ribulose-1,5-diphosphate carboxylase. All this evidence suggests the ability of T. roseopersicina to change from phototrophy to aerobic chemolithoautotrophy.