Single mRNA molecules demonstrate probabilistic movement in living mammalian cells

Cytoplasmic mRNA movements ultimately determine the spatial distribution of protein synthesis. Although some mRNAs are compartmentalized in cytoplasmic regions, most mRNAs, such as housekeeping mRNAs or the poly-adenylated mRNA population, are believed to be distributed throughout the cytoplasm. The general mechanism by which all mRNAs may move, and how this may be related to localization, is unknown. Here, we report a method to visualize single mRNA molecules in living mammalian cells, and we report that, regardless of any specific cytoplasmic distribution, individual mRNA molecules exhibit rapid and directional movements on microtubules. Importantly, the beta-actin mRNA zipcode increased both the frequency and length of these movements, providing a common mechanistic basis for both localized and nonlocalized mRNAs. Disruption of the cytoskeleton with drugs showed that microtubules and microfilaments are involved in the types of mRNA movements we have observed, which included complete immobility and corralled and nonrestricted diffusion. Individual mRNA molecules switched frequently among these movements, suggesting that mRNAs undergo continuous cycles of anchoring, diffusion, and active transport.     

Heterologous protein production capacity of mammalian cells cultivated as monolayers and microtissues

A precise understanding of processes managing heterologous protein production in vitro and in vivo is essential for the manufacture of sophisticated biopharmaceuticals as well as for future gene therapy and tissue engineering initiatives. Capitalizing on the gravity-enforced self-assembly of monodispersed cells into coherent (multicellular) microtissues we studied heterologous protein production of microtissues and monolayers derived from cell lines and primary cells engineered/transduced for (i) constitutive, (ii) proliferation-controlled, (iii) macrolide-, or (iv) gas-inducible expression of the human placental secreted alkaline phosphatase (SEAP) and of the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY). Specific productivity of cells assembled in microtissues was up to 20-fold higher than isogenic monolayer cultures. Diffusion across microtissues could be further increased by HUVEC-mediated vascularization. As well as higher specific protein productivities, microtissues were also more efficient than monolayer cultures in assembling transgenic lentiviral particles. Our results showed that mammalian cells embedded in a tissue-like three-dimensional (3D) microenvironment exhibit increased production capacity. This observation should be considered for gene therapy and tissue engineering scenarios as well as for biopharmaceutical manufacturing.     

Clonogenicity of mammalian cells in hybrid spheroids a new assay method

Summary A new in vitro method for determining the clonogenicity of mammalian cells in culture is described. The method is based on packaging clonogens into agglomerates of non-proliferating, but metabolically active, HeLa cells. These agglomerates, termed hybrid spheroids, provide an in vivo-like environment for entrapped test cells, offering a realistic system for prospective tumor control studies. Clonogenicity is determined by varying the number of test cells per hybrid spheroid so that some, but not all, spheroids give rise to macrocolonies. From the fraction of non-colony forming spheroids, the average number of clonogens per spheroid can be calculated, and the survival of irradiated test cells determined. In this fashion survival curves were obtained for HeLa, B-16 and HEp3 cells which corresponded to survival curves obtained in the conventional manner. The clonogenicity of cells, derived from a human maxillar melanoma surgical specimen was also determined by the hybrid spheroid method. With this method, plating efficiency increased in those cells which normally plate poorly, such as tumor cells, thus enabling survival measurements when this is not practical using conventional methods.     

A procedure to introduce protein molecules into living mammalian cells

Although several methods are now available by which to introduce macromolecules into cultured living mammalian cells, each has limitations on its adoption as a general means, for a variety of purposes. We describe here a simple procedure to introduce protein molecules into various living mammalian cells. This procedure is based upon the finding that mammalian cells, after exposure to a low concentration of a phospholipid (L-alpha-lysophosphatidylcholine) in the presence of high (hypertonic) concentrations of glycerol became permeable to protein molecules and that a significant portion of the exposed cells regain their viability following incubation in the appropriate growth medium. We have demonstrated that diphtheria toxin (A fragment), horseradish peroxidase and antibodies against SV40 T-antigens are incorporated into living mouse erythroleukemia (Friend) cells, baby hamster kidney (BHK) cells and mouse fibroblasts (C3H), respectively. The volume introduced into a single cell (mouse Friend cells) is approx. 3 X 10(-15) liter, which is comparable to those with other systems. Parameters affecting permeability to protein molecules and viability of the treated cells were also investigated with these and other cell lines.     

Lac operator repeats generate a traceable fragile site in mammalian cells

One limitation for the study of chromosomal fragile sites is that they must be studied on metaphase spreads, after the breakage. We show here that bacterial lac operator (lacO) repeats are prone to spontaneous breakage, which when combined with a fluorescent lac repressor (lacR) has allowed us to track a fragile site through the cell cycle. By using this system, we show that Plk1-interacting checkpoint helicase (PICH) is already present at fragile sites during interphase, suggesting roles for this helicase beyond mitosis. In addition, we report that the oncogene Myc promotes the formation of anaphase bridges and micronuclei containing fragile-site sequences.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

Pools and protein synthesis in mammalian cells.

From the kinetics of incorporation into protein shown by four amino acids and one amino acid analogue in suspension cultured HeLa S-3 cells, two distinctly different patterns were observed under the same experimental conditions. An initial slow exponential incorporation followed by linear kinetics was characteristic of the two non-essential amino acids, glycine and proline, whereas the two essential amino acids studied, phenylalanine and leucine, showed linear kinetics of incorporation with no detectable delay. The analogue amino acid, p-fluorophenylalanine also showed immediate linear kinetics of incorporation. There was a poor correlation between the rate of formation of acid-soluble pools and incorporation kinetics. However, the rate of formation of the freely diffusible pool of amino acids correlated more closely with incorporation kinetics. The lack of direct involvement of the acid-soluble pool in protein synthesis was also demonstrated by pre-loading of pools before treatment of cells with labelled amino acids. The results partially support the hypothesis that precursor amino acids for protein synthesis come from the external medium rather than the acid-soluble pool, but suggest that the amino acid which freely diffuses into the cell from the external medium could also be the source.     

A new U6 small nuclear ribonucleoprotein-specific protein conserved between cis- and trans-splicing systems.

Spliceosomal U6 small nuclear RNA (snRNA) plays a central role in the pre-mRNA splicing mechanism and is highly conserved throughout evolution. Previously, a sequence element essential for both capping and cytoplasmic-nuclear transport of U6 snRNA was mapped in the 5'-terminal domain of U6 snRNA. We have identified a protein in cytoplasmic extracts of mammalian and Trypanosoma brucei cells that binds specifically to this U6 snRNA element. Competition studies with mutant and heterologous RNAs demonstrated the conserved binding specificity of the mammalian and trypanosomal proteins. The in vitro capping analysis of mutant U6 snRNAs indicated that protein binding is required but not sufficient for capping of U6 snRNA by a gamma-monomethyl phosphate. Through RNA affinity purification of mammalian small nuclear ribonucleoproteins (snRNPs), we detected this protein also in nuclear extract as a new specific component of the U6 snRNP but surprisingly not of the U4/U6 or the U4/U5/U6 multi-snRNP. These results suggest that the U6-specific protein is involved in U6 snRNA maturation and transport and may therefore be functionally related to the Sm proteins of the other spliceosomal snRNPs.