Relationship between Arp2/3 complex and the barbed ends of actin filaments at the leading edge of carcinoma cells after epidermal growth factor stimulation.

Using both light and high resolution electron microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension in mammary carcinoma cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulation and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100-200 nm of the plasma membrane). This is accompanied by an increase in filament density at the leading edge and a general decrease in filament length, with a specific loss of long filaments. Concomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throughout the entire cortical filament network extending 1-1.5 microm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be involved in initial generation of growing filaments. However, only a small subset of the complex present in the cortical network colocalizes near free barbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochemical microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.

An improved method for generating BAC DNA suitable for FISH.

Fluorescence in situ hybridization (FISH) is commonly used to identify chromosomal aberrations such as translocations, deletions, duplications, gene fusions, and aneuploidies. It relies on the hybridization of fluorescently labeled DNA probes onto denatured metaphase chromosomes or interphase nuclei. These probes are often generated from DNA sequences cloned within bacterial artificial chromosomes (BACs). Growing these BACs in adequate amounts for FISH can be demanding. We describe FISH performed with bacteriophage Phi29 DNA polymerase amplified BAC DNA. Generating this material required significantly smaller cultures and less time than standard methods. The FISH results obtained were comparable with those obtained from standard BAC DNA. We believe this method of BAC DNA generation is useful for the entire FISH community as it improves considerably on prior methods.

Epidermal growth factor receptor distribution during chemotactic responses.

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

A procedure for RT-PCR amplification of mRNAs on histological specimens.

Detection in combination with localization of low copy gene expression can be difficult to achieve. The use of reverse transcription PCR on tissue sections with a fluorescent marker provides localization of mRNA expression on a cellular level, and when combined with confocal microscopy and image analysis it also allows for an estimate of the relative intensity of fluorescence. A good example of the application of this method is the localization of nerve growth factor (NGF) mRNA expression in the inner ear. NGF through indirect tests appears to be present in this system, yet NGF mRNA could not be localized with in situ hybridization using radiolabeled riboprobes. Using fluorescent in situ RT-PCR, we can easily detect the presence of NGF mRNA and localize it to specific cell types within the maturing inner ear.

Glial cell abnormalities in the CNS of the carbonic anhydrase II deficient mutant mouse.

The Car-2n/Car-2n mutant mouse is entirely lacking in carbonic anhydrase II (CA), which is normally found in myelin and glial cells in the CNS. CNS tissue from CA-deficient mutant mice was examined to see whether abnormalities could be detected which might help in understanding the function(s) of CA in the normal brain. Analyses of myelin yields and myelin proteins showed no differences between mutants and normal littermates. To visualize the oligodendrocytes and astrocytes in tissue sections, immunocytochemical staining was performed using antibodies against the Pi form of glutathione-S-transferase and glial-fibrillary acidic protein, respectively. In both gray and white matter from the mutants' brains the oligodendrocytes appeared to be shrinking and, possibly, degenerating. Hypertrophy of astrocytes occurred in the white matter, and the astrocytes in gray matter appeared swollen. It is suggested that imbalances in the HCO3-/CO2 ratios in various glial-cell compartments may produce abnormal distributions of water and ions in the brains of the CAII-deficient mice.

Organ-dependent variation of capsule thickness in Cryptococcus neoformans during experimental murine infection.

In studies of murine infection, the capsule thickness of Cryptococcus neoformans varied depending on the organ. The relative order of thickness was as follows: lung > brain > in vitro isolates. The differences in capsule thickness suggest that there are organ-related differences in the expression of genes responsible for capsule thickness.

Quantification of ribonucleotide reductase expression in wild-type and hydroxyurea-resistant cell lines employing in situ reverse transcriptase polymerase chain reaction and a computerized image analysis system.

Ribonucleotide reductase (RR) is the enzyme responsible for converting nucleoside diphosphates to deoxynucleoside diphosphates, ensuring a balanced supply of deoxyribonucleotides for DNA synthesis. Expression of RR is tightly regulated, but it is affected by exogenous agents, such as hydroxyurea (HU), which inactivates the tyrosyl free radical on the small subunit of RR, R2. We have previously employed in situ reverse transcriptase (RT)-PCR to estimate expression of R2 in wild-type and HU-resistant human colon carcinoma cell lines and to correlate altered expression of R2 with changes in cell size and morphology. The current studies were undertaken to render this methodology more quantitative. Both wild-type and resistant cells were grown on partitioned glass slides and analyzed with in situ RT-PCR. Because both wild-type and resistant cells were analyzed under a single cover slip, protease digestion, reverse transcription, PCR, and color development were all performed under identical conditions. Images were analyzed with NIH Image 1.59 software. There was a highly significant correlation between expression of R2 and cell size for both sensitive and resistant cells (P = 0.0001, for both). When cell size was compared either with expression of R2 or cell shape, however, these correlated only in wild-type cells (P = 0.001 and 0.0001, respectively). These data demonstrate that normal cell growth in the unperturbed wild-type cell line was closely linked to expression of R2, whereas in the resistant variants which overexpress R2, these correlations were absent, suggesting that HU resistance is related to loss of linkage between R2 expression and cell growth and confirming previous data relating overexpression of R2 with multiple other changes in the cell growth repertoire. Thus, we have demonstrated for the first time a quantitative application of in situ RT-PCR.

Role of rRAB22b, an oligodendrocyte protein, in regulation of transport of vesicles from trans Golgi to endocytic compartments.

Intracellular membrane trafficking plays an essential role in the biogenesis and maintenance of myelin. Members of the Rab protein family are important components of the systems that regulate intracellular vesicle transport. We examine the function of rRab22b, a novel rat Rab protein cloned from an oligodendrocyte cDNA library, by visualizing and identifying in living Hela cells the organelles that contain rRab22b. Our results show that rRab22b is present in the trans Golgi/TGN and endocytic compartments. Trafficking of membranes from trans Golgi to endocytic compartments takes place via small tubulo vesicular organelles containing rRab22b. The formation of vesicles in the trans Golgi also appears to be regulated by rRab22b. Additionally, our results suggest that rRab22b controls the transport of vesicles from the trans Golgi to endocytic compartments that localize in oligodendrocyte processes. That rRab22b is involved in the transport of certain proteins from trans Golgi to myelin is suggested by the evidence that certain proteins being targeted to the plasma membrane are first transported from trans Golgi to endocytic compartments.

Three-dimensional organization of rat hepatocyte cytoskeleton: relation to the asialoglycoprotein endocytosis pathway.

Analysis by confocal microscopy has revealed features of the microtubule network of rat hepatocytes in culture, establishing the three-dimensional disposition of the microtubule-based cytoskeleton, its relation to the actin-based cytoskeleton and to ligand-containing endosomes during receptor-mediated endocytosis and the alterations in its structure and disposition by the microtubule pertubant, Taxol. By co-localization studies, we have been able to demonstrate that the microtubules have a significant role in receptor-mediated endocytosis of asialoglycoproteins in this cell. Asialoorosomucoid-containing endosomes attach to widely spaced arrays of microtubules running under the baso-lateral surface of the hepatocytes 5-15 minutes after the initiation of endocytosis and then travel along microtubule paths to become concentrated with microtubules near the centrosome and at bile canaliculi after 30-60 minutes of receptor-mediated endocytosis. Receptor-mediated endocytosis is affected, but not abolished by Taxol, which inhibits the rate of asialoorosomucoid degradation at the same concentrations as those that disrupt microtubule and cytoplasmic dynein distribution, and that prevent the concentration of endosomes centrally. The results support suggestions that asialoorosomucoid-containing endosomes are captured by microtubules just below the actin layer at the cell periphery and these are actively transported centrally along microtubules, possibly by cytoplasmic dynein, so that the concentration of endosomes near the centrosome, and the subsequent efficient lysosomal degradation of ligand, are consequences of the confluence of microtubules in this region.

Mycobacterial infection of macrophages results in membrane-permeable phagosomes.

Cell-mediated immunity is critical for host resistance to tuberculosis. T lymphocytes recognizing antigens presented by the major histocompatibility complex (MHC) class I and class II molecules have been found to be necessary for control of mycobacterial infection. Mice genetically deficient in the generation of MHC class I and class Ia responses are susceptible to mycobacterial infection. Although soluble protein antigens are generally presented by macrophages to T cells through MHC class II molecules, macrophages infected with Mycobacterium tuberculosis or bacille Calmette-Guerin have been shown to facilitate presentation of ovalbumin through the MHC class I presentation pathway via a TAP-dependent mechanism. How mycobacteria, thought to reside within membrane-bound vacuoles, facilitate communication with the cytoplasm and enable MHC class I presentation presents a paradox. By using confocal microscopy to study the localization of fluorescent-tagged dextrans of varying size microinjected intracytoplasmically into macrophages infected with bacille Calmette-Guerin expressing the green fluorescent protein, molecules as large as 70 kilodaltons were shown to gain access to the mycobacterial phagosome. Possible biological consequences of the permeabilization of vacuolar membranes by mycobacteria would be pathogen access to host cell nutrients within the cytoplasm, perhaps contributing to bacterial pathogenesis, and access of microbial antigens to the MHC class I presentation pathway, contributing to host protective immune responses.