High-aperture cryogenic light microscopy.

We report here the development of instruments and protocols for carrying out high numerical aperture immersion light microscopy on cryogenic specimens. Imaging by this modality greatly increases the lifetimes of fluorescence probes, including those commonly used for protein localization studies, while retaining the ability to image the specimen with high fidelity and spatial resolution. The novel use of a cryogenic immersion fluid also minimizes the refractive index mismatch between the sample and lens, leading to a more efficient coupling of the light from the sample to the image forming system. This enhancement is applicable to both fluorescence and transmitted light microscopy techniques. The design concepts used for the cryogenic microscope can be applied to virtually any existing light-based microscopy technique. This prospect is particularly exciting in the context of 'super-resolution' techniques, where enhanced fluorescence lifetime probes are especially useful. Thus, using this new modality it is now possible to observe dynamic events in a live cell, and then rapidly vitrify the specimen at a specific time point prior to carrying out high-resolution imaging. The techniques described can be used in conjunction with other imaging modalities in correlated studies. We have also developed instrumentation to perform cryo-light imaging together with soft X-ray tomography on the same cryo-fixed specimen as a means of carrying out high content, quantifiable correlated imaging analyses. These methods are equally applicable to correlated light and electron microscopy of frozen biological objects.

High resolution protein localization using soft X-ray microscopy.

Soft X-ray microscopes can be used to examine whole, hydrated cells up to 10 microm thick and produce images approaching 30 nm resolution. Since cells are imaged in the X-ray transmissive "water window", where organic material absorbs approximately an order of magnitude more strongly than water, chemical contrast enhancement agents are not required to view the distribution of cellular structures. Although living specimens cannot be examined, cells can be rapidly frozen at a precise moment in time and examined in a cryostage, revealing information that most closely approximates that in live cells. In this study, we used a transmission X-ray microscope at photon energies just below the oxygen edge (lambda = 2.4 nm) to examine rapidly frozen mouse 3T3 cells and obtained excellent cellular morphology at better than 50 nm lateral resolution. These specimens are extremely stable, enabling multiple exposures with virtually no detectable damage to cell structures. We also show that silver-enhanced, immunogold labelling can be used to localize both cytoplasmic and nuclear proteins in whole, hydrated mammary epithelial cells at better than 50 nm resolution. The future use of X-ray tomography, along with improved zone plate lenses, will enable collection of better resolution (approaching 30 nm), three-dimensional information on the distribution of proteins in cells.

Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP.

We examined the spatial and temporal control of actin assembly in living Xenopus eggs. Within minutes of egg activation, dynamic actin-rich comet tails appeared on a subset of cytoplasmic vesicles that were enriched in protein kinase C (PKC), causing the vesicles to move through the cytoplasm. Actin comet tail formation in vivo was stimulated by the PKC activator phorbol myristate acetate (PMA), and this process could be reconstituted in a cell-free system. We used this system to define the characteristics that distinguish vesicles associated with actin comet tails from other vesicles in the extract. We found that the protein, N-WASP, was recruited to the surface of every vesicle associated with an actin comet tail, suggesting that vesicle movement results from actin assembly nucleated by the Arp2/3 complex, the immediate downstream target of N-WASP. The motile vesicles accumulated the dye acridine orange, a marker for endosomes and lysosomes. Furthermore, vesicles associated with actin comet tails had the morphological features of multivesicular endosomes as revealed by electron microscopy. Endosomes and lysosomes from mammalian cells preferentially nucleated actin assembly and moved in the Xenopus egg extract system. These results define endosomes and lysosomes as recruitment sites for the actin nucleation machinery and demonstrate that actin assembly contributes to organelle movement. Conversely, by nucleating actin assembly, intracellular membranes may contribute to the dynamic organization of the actin cytoskeleton.

Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.

Examination of the subcellular localization of Dishevelled (Dsh) in fertilized Xenopus eggs revealed that Dsh is associated with vesicle-like organelles that are enriched on the prospective dorsal side of the embryo after cortical rotation. Dorsal enrichment of Dsh is blocked by UV irradiation of the vegetal pole, a treatment that inhibits development of dorsal cell fates, linking accumulation of Dsh and specification of dorsal cell fates. Investigation of the dynamics of Dsh localization using Dsh tagged with green fluorescent protein (Dsh-GFP) demonstrated that Dsh-GFP associates with small vesicle-like organelles that are directionally transported along the parallel array of microtubules towards the prospective dorsal side of the embryo during cortical rotation. Perturbing the assembly of the microtubule array with D(2)O, a treatment that promotes the random assembly of the array and the dorsalization of embryos, randomizes translocation of Dsh-GFP. Conversely, UV irradiation of the vegetal pole abolishes movement of Dsh-GFP. Finally, we demonstrate that overexpression of Dsh can stabilize beta-catenin in Xenopus. These data suggest that the directional translocation of Dsh along microtubules during cortical rotation and its subsequent enrichment on the prospective dorsal side of the embryo play a role in locally activating a maternal Wnt pathway responsible for establishing dorsal cell fates in Xenopus.

Tissue phenotype depends on reciprocal interactions between the extracellular matrix and the structural organization of the nucleus.

What determines the nuclear organization within a cell and whether this organization itself can impose cellular function within a tissue remains unknown. To explore the relationship between nuclear organization and tissue architecture and function, we used a model of human mammary epithelial cell acinar morphogenesis. When cultured within a reconstituted basement membrane (rBM), HMT-3522 cells form polarized and growth-arrested tissue-like acini with a central lumen and deposit an endogenous BM. We show that rBM-induced morphogenesis is accompanied by relocalization of the nuclear matrix proteins NuMA, splicing factor SRm160, and cell cycle regulator Rb. These proteins had distinct distribution patterns specific for proliferation, growth arrest, and acini formation, whereas the distribution of the nuclear lamina protein, lamin B, remained unchanged. NuMA relocalized to foci, which coalesced into larger assemblies as morphogenesis progressed. Perturbation of histone acetylation in the acini by trichostatin A treatment altered chromatin structure, disrupted NuMA foci, and induced cell proliferation. Moreover, treatment of transiently permeabilized acini with a NuMA antibody led to the disruption of NuMA foci, alteration of histone acetylation, activation of metalloproteases, and breakdown of the endogenous BM. These results experimentally demonstrate a dynamic interaction between the extracellular matrix, nuclear organization, and tissue phenotype. They further show that rather than passively reflecting changes in gene expression, nuclear organization itself can modulate the cellular and tissue phenotype.

Reciprocal interactions between beta1-integrin and epidermal growth factor receptor in three-dimensional basement membrane breast cultures: a different perspective in epithelial biology.

Anchorage and growth factor independence are cardinal features of the transformed phenotype. Although it is logical that the two pathways must be coregulated in normal tissues to maintain homeostasis, this has not been demonstrated directly. We showed previously that down-modulation of beta1-integrin signaling reverted the malignant behavior of a human breast tumor cell line (T4-2) derived from phenotypically normal cells (HMT-3522) and led to growth arrest in a three-dimensional (3D) basement membrane assay in which the cells formed tissue-like acini (14). Here, we show that there is a bidirectional cross-modulation of beta1-integrin and epidermal growth factor receptor (EGFR) signaling via the mitogen-activated protein kinase (MAPK) pathway. The reciprocal modulation does not occur in monolayer (2D) cultures. Antibody-mediated inhibition of either of these receptors in the tumor cells, or inhibition of MAPK kinase, induced a concomitant down-regulation of both receptors, followed by growth-arrest and restoration of normal breast tissue morphogenesis. Cross-modulation and tissue morphogenesis were associated with attenuation of EGF-induced transient MAPK activation. To specifically test EGFR and beta1-integrin interdependency, EGFR was overexpressed in nonmalignant cells, leading to disruption of morphogenesis and a compensatory up-regulation of beta1-integrin expression, again only in 3D. Our results indicate that when breast cells are spatially organized as a result of contact with basement membrane, the signaling pathways become coupled and bidirectional. They further explain why breast cells fail to differentiate in monolayer cultures in which these events are mostly uncoupled. Moreover, in a subset of tumor cells in which these pathways are misregulated but functional, the cells could be "normalized" by manipulating either pathway.

Structural protein 4.1 in the nucleus of human cells: dynamic rearrangements during cell division.

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function.

Reversion of the malignant phenotype of human breast cells in three-dimensional culture and in vivo by integrin blocking antibodies.

In a recently developed human breast cancer model, treatment of tumor cells in a 3-dimensional culture with inhibitory beta1-integrin antibody or its Fab fragments led to a striking morphological and functional reversion to a normal phenotype. A stimulatory beta1-integrin antibody proved to be ineffective. The newly formed reverted acini re-assembled a basement membrane and re-established E-cadherin-catenin complexes, and re-organized their cytoskeletons. At the same time they downregulated cyclin D1, upregulated p21(cip,wat-1), and stopped growing. Tumor cells treated with the same antibody and injected into nude mice had significantly reduced number and size of tumors in nude mice. The tissue distribution of other integrins was also normalized, suggesting the existence of intimate interactions between the different integrin pathways as well as adherens junctions. On the other hand, nonmalignant cells when treated with either alpha6 or beta4 function altering antibodies continued to grow, and had disorganized colony morphologies resembling the untreated tumor colonies. This shows a significant role of the alpha6/beta4 heterodimer in directing polarity and tissue structure. The observed phenotypes were reversible when the cells were disassociated and the antibodies removed. Our results illustrate that the extracellular matrix and its receptors dictate the phenotype of mammary epithelial cells, and thus in this model system the tissue phenotype is dominant over the cellular genotype.

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in beta-catenin that are modulated by the Wnt signaling pathway.

Eggs of Xenopus laevis undergo a postfertilization cortical rotation that specifies the position of the dorso-ventral axis and activates a transplantable dorsal-determining activity in dorsal blastomeres by the 32-cell stage. There have heretofore been no reported dorso-ventral asymmetries in endogenous signaling proteins that may be involved in this dorsal-determining activity during early cleavage stages. We focused on beta-catenin as a candidate for an asymmetrically localized dorsal-determining factor since it is both necessary and sufficient for dorsal axis formation. We report that beta-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with beta-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32-cell stages. We then investigated which potential signaling factors and pathways are capable of modulating the steady-state levels of endogenous beta-catenin. Steady-state levels and nuclear accumulation of beta-catenin increased in response to ectopic Xenopus Wnt-8 (Xwnt-8) and to the inhibition of glycogen synthase kinase-3, whereas neither Xwnt-5A, BVg1, nor noggin increased beta-catenin levels before the mid-blastula stage. As greater levels and nuclear accumulation of beta-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, our data suggest that early asymmetries in beta-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. Our data further support the hypothesis that these dorso-ventral differences in beta-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3.

Microtubule-mediated transport of organelles and localization of beta-catenin to the future dorsal side of Xenopus eggs.

The dorsal-ventral axis in frog embryos is specified during the first cell cycle, when the cortex rotates relative to the cytoplasmic core along parallel microtubules associated with the core. Cytoplasmic transfer experiments suggest that dorsal determinants are transported 90 degrees from the vegetal pole to the dorsal equator, even though the cortex rotates only 30 degrees. Here we show that, during rotation, small endogenous organelles are rapidly propelled along the subcortical microtubules toward the future dorsal side and that fluorescent carboxylated beads injected into the vegetal pole are transported at least 60 degrees toward the equator. We also show that deuterium oxide, which broadens the zone of dorsalization even though it reduces the extent of rotation and is known to randomize the microtubules, also randomizes the direction of organelle transport. Moreover, beta-catenin, a component of the Wnt signaling pathway that possesses dorsalizing activity in Xenopus, colocalizes with subcortical microtubules at the dorsal side of the egg at the end of rotation. We propose that cortical rotation functions to align subcortical microtubules, which then mediate the transport of dorsal determinants toward their plus ends on one side of the egg.

Confocal microscopy analysis of living Xenopus eggs and the mechanism of cortical rotation.

The dorsoventral body axis in amphibian embryos is established by a rotation of the outer cortex relative to the inner cytoplasmic core. This cortical rotation depends on microtubules and is correlated with a parallel array of microtubules just inside the vegetal cortex. Since the parallel array moves with the inner cytoplasm and most of its microtubules are oriented with their plus ends facing the direction of cortical movement, it has been suggested that plus end-directed motor molecules attached to the cortex drive the rotation by moving along microtubules of the parallel array. Using an inverted confocal microscope to examine living eggs, however, we found that rotation movements precede the formation of a detectable parallel array at the vegetal pole, that the parallel array consists of multiple layers of microtubules at depths ranging from 4 to 8 microns inside the plasma membrane and that the velocity of rotation is immobilized eggs increases with depth in this region. These findings suggest that (1) early cytoplasmic movements are due to something other than the fully formed parallel array and (2) the motor molecules responsible for the bulk of the rotation movement are not restricted to a monolayer at the subcortical interface but may be distributed throughout the parallel array, perhaps causing microtubules to slide along other microtubules by a mechanism similar to that seen in cilia and eukaryotic flagella.

Novel cytoskeletal elements in mammalian eggs are composed of a unique arrangement of intermediate filaments.

Mammalian eggs and embryos contain a major network of specialized cytoskeletal components known as 'sheets' that have not been identified in any other cell type. Although eggs from at least seven different mammalian species have been shown to contain these cytoskeletal structures, embedment-free electron microscopic analysis of these eggs revealed that two basic forms of cytoskeletal sheets exist, a solid, planar type of sheet typical of hamster and rat eggs and a fibrous sheet typical of mouse, porcine, bovine, canine, and human eggs. In this study we have investigated the structural composition of the fibrous type of sheet in mouse eggs by employing biochemical approaches as well as two forms of ultrastructural analyses including: (1) analysis of thick, resin-embedded specimens using an intermediate voltage electron microscope (IVEM); (2) analysis of replicas from quick-frozen, deep-etched specimens. Our results indicate that the sheets of mouse eggs and preimplantation embryos are composed of cylindrical bundles of 10-11 nm filaments, with each of these filaments held in register by periodically arranged crossbridges spaced 23-25 nm apart. This sheet substructure of filaments and crossbridges is covered by a particulate material which can be removed by non-ionic detergent. Immunoelectron microscopic analysis of mouse eggs demonstrates that sheets bind antibodies to keratin and to a small extent, actin, but do not bind antibodies to vimentin or tubulin. Confirmation that keratin exists in these eggs was obtained by electrophoretic separation and one- and two-dimensional Western blot analysis demonstrating the existence of keratin types 5, 6, 8, 16, and type Z. The low abundancy of keratin type 8 compared to other keratin types explains the difficulties other investigators have had identifying intermediate filaments in mammalian embryos since most investigators have used antibodies directed specifically against keratin type 8 or its pair keratin type 18. Examination of compacted mouse embryos reveals that the filamentous framework of sheets disassembled and established close contact with the basolateral plasma membrane and the nucleus. However, sheets at the apical plasma membrane of blastomeres attach to the membrane but remain intact. Based on our biochemical and ultrastructural data, the fibrous sheets of mouse eggs appear to be cytoskeletal structures comparable to the solid, planar sheets of the Syrian hamster egg and probably serve similar function(s) in eggs and embryos of several mammalian species.

Desmosome differentiation during epidermal cornification: new observations obtained from intermediate voltage electron microscopy.

Human epidermis was examined under 400-kV intermediate voltage electron microscopy using epon sections cut 5-8 times thicker than usual ultrathin sections. Asymmetry of desmosomal structures occurred as cells became cornified. Electron-dense proteins deposited at the inner leaflet of the plasma membrane of corneocytes do not extend into the desmosomal area, which connects to granular cells. Stereo micrographs revealed the existence of two different cellular elements at the cell surface confirming that the plasma membrane first thickened in areas without desmosomes. Examination of the three-dimensional nature of desmosomes and keratin-filament aggregation without serial sectioning and/or selecting a strict angle of the tissues will allow us to extend our ultrastructural knowledge.

A new technique for isolation and visualization of the Xenopus egg cortex reveals a complex cytoskeleton.

This paper demonstrates the first images of the isolated cortex of the Xenopus laevis egg viewed as a whole mount. The cortex was isolated by mechanically restraining it in a folding hexagonal mesh electron microscopy grid, dried by passage through the carbon dioxide critical point, and viewed in an intermediate voltage electron microscope at 400 kV. Using this approach, we have obtained new views of the Xenopus egg cortex that demonstrate a complex cytoskeleton. Stereo micrographs demonstrate three-dimensional views of the cortical organelles and their interaction with the cytoskeleton. These images demonstrate the presence of a dense meshwork of filaments located just beneath the plasma membrane, referred to as the submembranous cytoskeleton. In addition, there is an array of filaments interconnecting the organelles and securing them to the submembranous cytoskeleton and plasma membrane. The development of this technique for visualization of the egg cortex will facilitate further analysis of the role of the cortical cytoskeleton in fertilization and early embryogenesis.

Cytoskeletal sheets of mammalian eggs and embryos: a lattice-like network of intermediate filaments.

Mammalian eggs and embryos possess a major cytoskeletal network composed of large planar "sheets" distributed throughout the cytoplasm. Cytoskeletal sheets are found neither in mammalian somatic cells nor in eggs or embryos of non-mammals. In this study, we have investigated the structural composition of the sheets in eggs and embryos of the golden Syrian hamster by (1) analysis of replicas from quick-frozen, deep-etched specimens, (2) analysis of thick, resin-embedded specimens using an intermediate voltage electron microscope (IVEM), (3) laser diffraction of EM images, (4) differential extraction with detergents, and (5) immunocytochemistry. Our results indicate that each sheet is composed of two closely apposed arrays of 10-nm filaments. Each filament within an array is held in register with its neighbor by lateral cross-bridges and the two parallel arrays of filaments are interconnected by periodic cross-bridges about 20 nm in length. Laser diffraction of negatives from IVEM images indicates that each array is composed of fibers that form a square lattice, and the two arrays are positioned in register by cross-bridges forming a single sheet. This lattice forms the skeleton of the sheets which is covered with a tightly packed layer of particulate material. By incubation in media containing different ratios of mixed-micelle detergents, it is possible to remove components sequentially from the sheets and to extract the particulate material. Immunocytochemical localization demonstrates that the sheets bind antibodies to keratin, and to a small extent actin, but do not bind antibodies to vimentin or tubulin. Examination of sheets within embryos at the time of embryonic compaction demonstrates that the sheets begin to fragment and disassemble in regions of blastomeres where desmosomes form, but undergo no structural alterations in interior and basal surfaces of the blastomeres. In regions of blastomere-blastomere contact the sheets fragment and associate with granules resembling keratohyalin granules found in keratinocytes.

Localization of a chymotrypsin-like protease to the perivitelline space of Xenopus laevis eggs.

A chymotrypsin-like protease is released from Xenopus laevis eggs at activation and is involved in conversion of the vitelline envelope to the fertilization envelope. To localize this enzyme in unactivated and activated eggs, we used the synthetic peptide substrate succinylalanylalanylprolylphenylalanyl-4-methoxy-2-naphthylamide whose product can be visualized using transmission electron microscopy. Protease product was localized within the perivitelline space of unactivated eggs, appearing as strings of beads. No protease activity was detected in activated eggs, which is consistent with the observation that the protease is released from the egg at activation.

Cortical membrane-trafficking during the meiotic resumption of Xenopus laevis oocytes.

Changes in the organization of membranous structures in the amphibian oocyte cortex were studied during the process of progesterone-induced meiotic resumption. Progesterone treatment of Xenopus laevis oocytes induced short term and longer term changes in the cortical membranes. In the short term, progesterone induced a burst of endocytosis mediated through coated pits and coated vesicles. Immuno-electron-microscopic localization of progesterone suggested that the progesterone receptor, bound to its ligand, is endocytosed during progesterone-induced endocytosis. Also demonstrated was the existence of a cisternal membrane network, referred to as the primordial cortical endoplasmic reticulum, which surrounds portions of the cortical granules in oocytes. The primordial cortical endoplasmic reticulum is more highly developed in the animal hemisphere than the vegetal hemisphere. Over the long term, during the meiotic resumption, more membrane is recruited into this network to form the cortical endoplasmic reticulum observed by others in the metaphase II egg. This evidence demonstrates that the cortex serves as a site for dynamic changes in membrane organization and that the most extensive changes occur in the animal hemisphere. These data support previous observations that the animal hemisphere is better structured for sperm penetration than is the vegetal hemisphere.