Previous Electron Microscopic Studies Research Articles

An immunocytochemical, cytosol, and ultrastructural study of tissue ferritin in breast carcinoma

Increased levels of serum ferritin in breast carcinoma is well known, however, its exact source has been disputed. Weinstein et al found six times the ferritin concentration in malignant tissue as compared to benign tissue. The anaplastic tumors had the highest ferritin concentrations, suggesting that the major site of the increased ferritin was the malignant epithelium. We found in a previous cytosolic and electron microscopic study of tissue ferritin in breast carcinoma that the malignant epithelium is almost certainly the source of the increased ferritin.To better define and expand this study and increase our knowledge of the role of transferrin receptors and iron metabolism in breast cancer, we began an immunocytochemical study of tissue ferritin to compliment our cytosol and ultrastructural observations. This was done with a mouse antihuman ferritin monoclonal antibody. To date 92 specimens have been examined by all three techniques. Immunocytochemical tissue slides showed intense staining of the cytoplasm with immunoperoxidase, and the intensity of the cytoplasmic staining correlated with the cytosolic concentrations and the electron microscopic findings.

Morphological types of neurons located at taste‐responsive sites in the solitary nucleus of the hamster

HRP histochemistry and microelectrode mapping were combined to study the sizes, shapes, and orientations of neuronal cell bodies and dendrites located at sites of taste-elicited single unit activity in the nucleus of the solitary tract (NST). Cells responding to sapid stimulation of the anterior tongue were extracellularly recorded using micropipettes containing HRP. Iontophoretic injection of the marker at the recording sites resulted in small (50-200 microns diameter) opaque zones bordered by a small number (2-15) of neurons with Golgi-like filling of their cell bodies, dendrites, and to some extent, their axons. The cell bodies were near (50-250 microns) the injection sites into which they sent labelled dendrites. Two broad categories of neurons were typically filled. Elongate cells had oval- to spindle-shaped cell bodies oriented mediolaterally. Two primary dendrites extended 100-300 microns from the cell body, one medially and one laterally, and branched within a cylindrical dendritic field oriented mediolaterally. A minority of the HRP-filled elongate cells had unusually long rostrally or caudally directed dendritic branches. Stellate cells had oval, round, triangular, or polygonal cell bodies and 3-5 primary dendrites coursing 200-300 microns in all directions and branching as unoriented, spheroidal fields. A minority of stellate cells had relatively unbranched wavy dendrites, resembling tentacles, while others had unusually small cell bodies (10-15 microns diameter), small dendrites, and locally arborizing axons. Of 151 labelled cells, all but 12 were remarkably confined to the rostral NST. Nearly 90% were concentrated in the rostral central cytoarchitectonic subdivision, where stellate cells predominated, or in the rostral lateral subdivision, where elongate cells predominated. These morphological types of neurons, filled at neurophysiological recording sites, are compared with cell types identified in previous light and electron microscopic studies of the cytoarchitecture, connections, and synaptic organization of the gustatory NST.

Phakomatous Choristoma (Zimmerman's tumor): Immunohistochemical Confirmation of Lens-specific Proteins

Phakomatous choristoma is a rare, congenital, ocular adnexal tumor that is presumed to be of lenticular anlage based on light and electron microscopy. The authors performed immunohistochemistry using standard commercially available antibodies against vimentin, S-100 protein, and several cytokeratins on a phakomatous choristoma that was excised from the right lower eyelid of a 10-week-old white boy. In addition, a battery of antibodies against lens-specific proteins, including alpha, beta, and gamma crystallins, was used. The tumor cells showed intense immunoreactivity for all lens-specific proteins tested. The epithelial cells of the phakomatous choristoma stained positively for S-100 protein and vimentin, the intermediate filament normally found in lens epithelial cells. Keratin markers were negative. The results of immunohistochemistry indicate that the cells of phakomatous choristoma synthesize several types of lens-specific proteins. Complementing previous light and electron microscopic studies, these data strongly support Zimmerman's conclusion that this pediatric adnexal tumor is a choristoma of lenticular anlage.

DiOC6 staining reveals organelle structure and dynamics in living yeast cells

When present at low concentrations, the fluorescent lipophilic dye, DiOC6, stains mitochondria in living yeast cells [Pringle et al.: Methods in Cell Biol. 31:357-435, 1989; Weisman et al.: Proc. Natl. Acad. Sci. U.S.A. 87:1076-1080, 1990]. However, we found that the nuclear envelope and endoplasmic reticulum were specifically stained if the dye concentration was increased or if certain respiratory-deficient yeast strains were examined. The quality of nuclear envelope staining with DiOC6 was sufficiently sensitive to reveal alterations in the nuclear envelope known as karmellae. These membranes were previously apparent only by electron microscopy. At the high dye concentrations required to stain the nuclear envelope, wild-type cells could no longer grow on non-fermentable carbon sources. In spite of this effect on mitochondrial function, the presence of high dye concentration did not adversely affect cell viability or general growth characteristics when strains were grown under standard conditions on glucose. Consequently, time-lapse confocal microscopy was used to examine organelle dynamics in living yeast cells stained with DiOC6. These in vivo observations correlated very well with previous electron microscopic studies, including analyses of mitochondria, karmellae, and mitosis. For example, cycles of mitochondrial fusion and division, as well as the changes in nuclear shape and position that occur during mitosis, were readily imaged in time-lapse studies of living DiOC6-stained cells. This technique also revealed new aspects of nuclear disposition and interactions with other organelles. For example, the nucleus and vacuole appeared to form a structurally coupled unit that could undergo coordinated movements. Furthermore, unlike the general view that nuclear movements occur only in association with division, the nucleus/vacuole underwent dramatic migrations around the cell periphery as cells exited from stationary phase. In addition to the large migrations or rotations of the nucleus/vacuole, DiOC6 staining also revealed more subtle dynamics, including the forces of the spindle on the nuclear envelope during mitosis. This technique should have broad application in analyses of yeast cell structure and function.

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHAm). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHAm and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHAm and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHAm and PHB accumulating strains have shown that PHAm and PHB granules can be distinguished from each other: PHAm granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHAm and PHB chains were stored in separate granules. To be able to determine whether the separation of PHAm and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHAm granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHAm and PHB, the resulting polymer chains are segregated completely and stored in separate granules.

Progressive subcortical vascular encephalopathy of the Binswanger type (PSVE)

Histopathological diffuse pallor of the cerebral white matter on myelin staining is a characteristic feature of progressive subcortical vascular encephalopathy of the Binswanger type (PSVE). However, the reason for the diffuse pallor was unclear. In a previous electron microscopic study the author reported that the number of nerve fibers per unit area of the frontal white matter was significantly less in PSVE than in the controls. The white matter pallor in PSVE is mainly related to the loss of nerve fibers there. Dementia in PSVE is probably related to the loss of nerve fibers in the cerebral white matter. The sum of the number of oligodendrocytes and astrocytes in the deep white matter in PSVE was approximately half of that in age-matched controls. The number of oligodendrocytes, which were defined as cells with small, round and solid nuclei, also decreased in PSVE being half of that in the deep white matter of the controls. These data suggest that the loss of oligodendrocytes and astrocytes plays a role in the process of nerve fibers loss in PSVE. Diffuse atherosclerotic lesions in the main stem of cerebral arteries and severe hyalinotic changes in the white matter arterioles are characteristic arterial lesions of PSVE. On the basis of severe arteriosclerosis in the cerebral arteries, it was speculated that repeated hypotension or greater variability of casual blood pressure could induce PSVE in the elderly hypertensives. To confirm the hypothesis, casual blood pressure in PSVE was retrospectively studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Extracellular Matrix Augments Mechanical Properties of Pseudopodia in the Carnivorous Foraminiferan Astrammina rara: Role in Prey Capture

The seemingly delicate, strand‐like pseudopodia of Astrammina rara, a carnivorous benthic foraminiferan, adhere to and withstand the rigorous movements of meiofaunal prey. Previous electron microscopic studies identified two novel structures that might account for the unusual tensile properties of these pseudopodia: 1) an extensive, coiled microtubule cytoskeleton and 2) a fibrous extracellular matrix vesting the pseudopodial surface. In the present study, we found that pseudopodial networks microsurgically removed from A. rara's cell body captured Artemia metanauplii as efficiently as intact organisms, and therefore used them to test the role of microtubules and extracellular matrix components in augmenting pseudopodial strength. Agents that specifically disassemble micro‐tubules (1 mM colchicine or 20 μM nocodazole) or generally disrupt pseudopodial integrity (heat, 10 mM formaldehyde, 1 mg/ml saponin) failed to inhibit prey capture. All of these treatments left the extracellular matrix intact as revealed by immunofluorescence and scanning electron microscopy. The elastic and tensile properties of the extracellular matrix, isolated by solubilization of pseudopodial cytonhsm using the nonionic detergent Triton X‐100, were similar to those of intact pseudopodial networks when assayed with calibrated microneedles or a flexible rubber substrate. These observations indicate that A. rara uses a fibrous extracellular matrix to augment cytoplasmic tensile properties.

HREM investigation of mesopore formation, aluminum migration in USY catalysts

The catalytic properties of ultrastable Y (USY) zeolite are directly influenced by the zeolite destruction which occurs during formation of USY and during subsequent hydrothermal treatment Previous transmission electron microscope (TEM) studies of hydrothermal aging of neat USY materials and also of USY cracking catalysts have shown 5-50nm defect domains, which were attributed to mesopores. Such features are characteristic of extended hydrothermal treatment. It has been suggested that these regions of zeolite destruction comprise the silica source for “healing” the tetrahedral site vacancies left by hydrothermal dealumination. The observations of both homogeneous and inhomogeneous distribution of these mesopores have been reported.

Molecular modeling of the domain structure of C9 of human complement by neutron and x-ray solution scattering

C9 is the most abundant component of the membrane attack complex of the complement system of immune defense. This is a typical mosaic protein with thrombospondin (TSR) and low density lipoprotein receptor (LDLr) domains at its N-terminus and an epidermal growth factor-like (EGF) domain at its C-terminus. Between these lies a perforin-like sequence. In order to define the arrangement in solution of these four moieties in C9, high-flux neutron and synchrotron X-ray solution scattering studies were carried out. The neutron radius of gyration RG at infinite contrast is 3.33 nm, and its cross-sectional RG (RXS) is 1.66 nm. Similar values were obtained by synchrotron X-ray scattering after allowance for radiation effects. Stuhrmann analyses showed that the neutron radial inhomogeneity of scattering density alpha is 35 X 10(-5) from the RG data and 16 X 10(-5) from the RXS data. These values are typical for soluble glycoproteins and show no evidence for the existence of any large hydrophobic surface patches on free C9 that might form contacts with lipids. Indirect transformation of the neutron and X-ray scattering curves into real space showed that C9 had a maximum dimension estimated at 12 +/- 2 nm, and this suggests that the lengths of 7-8 nm deduced from previous electron microscopy studies in vacuo are underestimated. Molecular modeling of the C9 scattering curves utilized small spheres in the Debye equation, in which the analyses were constrained by the known volumes of the four moieties of C9 and the known sizes of the TSR and EGF-like domains. The most likely models for C9 suggest that these four regions of C9 are arranged in a V-shaped structure, with an angle of 10 degrees between the two arms, each of length 11.1 nm. This structure has a more hydrophobic character between the two arms. The scattering model is fully consistent with hydrodynamic sedimentation data on C9. Similar V-shaped hydrodynamic models could be developed for C6, C7, C8, and C9 of complement. Such a compact structure is atypical of other multidomain complement proteins so far studied by solution scattering and is fully compatible with mechanisms in which C9 is postulated, on activation, to undergo a drastic unfolding of its domain structure and to expose a more hydrophobic surface which can be embedded into lipid bilayers.

Cryoelectron microscopy of mammalian pyruvate dehydrogenase complex.

Cryoelectron microscopy has been performed on frozen-hydrated pyruvate dehydrogenase complexes from bovine heart and kidney and on various subcomplexes consisting of the dihydrolipoyl transacetylase-based (E2) core and substoichiometric levels of the other two major components, pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3). The diameter of frozen-hydrated pyruvate dehydrogenase complex (PDC) is 50 nm, which is significantly larger than previously reported values. On the basis of micrographs of the subcomplexes, it is concluded that the E1 and E3 are attached to the E2-core complex by extended (4-6 nm maximally) flexible tethers. PDC constructed in this manner would probably collapse and appear smaller than its native size when dehydrated, as was the case in previous electron microscopy studies. The tether linking E1 to the core involves the hinge sequence located between the E1-binding and catalytic domains in the primary sequence of E2, whereas the tether linking E3 is probably derived from a similar hinge-type sequence in component X. Tilting of the E2-based cores and comparison with model structures confirmed that their overall shape is that of a pentagonal dodecahedron. The approximately 6 copies of protein X present in PDC do not appear to be clustered in one or two regions of the complex and are not likely to be symmetrically distributed.

Signet Ring Cell Basal Cell Carcinoma

A 63-year-old man presented with a signet ring cell basal cell carcinoma of the right infraorbital area. This is the third reported case of this rare variant of basal cell carcinoma characterized by tumor cells containing large, hyalinized, eccentric, intracytoplasmic inclusions that compress nuclei into crescent or ring-shaped forms. Antibodies to both high and low molecular weight cytokeratins were strongly positive, staining the inclusions in a uniform fashion. Vimentin and actin antibodies did not stain the inclusions. These results support previous electron microscopic studies that show the inclusions to be aggregates of intermediate filaments blending into tonofilaments at their periphery. Although speculative, the formation of signet ring cells does not appear to be a degenerative or necrotic phenomenon, but probably a peculiar aberrant form of individual cell keratinization.

The surface energy of metastable Al 3Li precipitates from coarsening kinetics

Small angle X-ray scattering has been used to measure the coarsening kinetics of metastable Al 3Li precipitates in a dilute Al Li binary alloy. The results are compared to previous transmission electron microscopy studies of similar alloys. Lifschitz-Slyozov-Wagner coarsening theory was invoked as a means of obtaining the α/Al 3Li interfacial energy. However, corrections accounting for effects due to finite volume fractions and limited solubility of Li in the precipitate phase are included. The surface energy was found to be ∼0.005J/m 2, significantly less than previously reported. A possible explanation for the discrepancy is discussed.

Division of astroblasts and oligodendroblasts in postnatal rodent brain: Evidence for separate astrocyte and oligodendrocyte lineages

What precursor cells are the source of the macroglia generated during postnatal development? In order to answer this question, we studied the expression of glial specific antigens in proliferating neuroglia in postnatal rodent brain and optic nerve. Immunocytochemistry using antibodies to oligodendrocyte (OL) specific markers (sulfatide and galactocerebroside) and an astrocyte (AS) specific marker (glial fibrillary acidic protein) was combined with thymidine autoradiography. During the first week of postnatal development when most ASs are being generated, one third to one half of the proliferating cells in the optic system are positive for glial fibrillary acidic protein after a 1 h injection of thymidine (Skoff, Dev. Biol., 139:149-168, 1990). During the second postnatal week when OLs are being generated, 30 to 100% of the proliferating cells in presumptive white matter tracts are sulfatide positive and at least 10% are galactocerebroside positive. This finding demonstrates that ASs and OLs divide during postnatal development. These results confirm previous electron microscopic autoradiographic studies showing that the vast majority of proliferating cells in postnatal rat optic nerve have the morphologic characteristics of differentiating ASs or OLs (Skoff, J. Comp. Neurol., 169:291-312, 1976). Since proliferating ASs (astroblasts) and OLs (oligodendroblasts) constitute the majority of the dividing cells at the time that ASs and OLs are being generated, these glioblasts must be the major source for the macroglia generated postnatally. The findings strongly suggest that separate lineages exist for ASs and OLs during postnatal development. There is no compelling in vivo evidence for a bipotential progenitor cell that generates the majority of OLs and certain ASs in postnatal rodent brain. There may, of course, be distinct lineages for the subtypes of ASs and possibly even for subtypes of OLs. We review the concepts of commitment and plasticity and apply these terms to glial differentiation. In situ, the presence of oligodendroblasts and astroblasts demonstrates the COMMITMENT of proliferating cells to a specific glial lineage during normal development. Culture conditions may provide an environment that permits proliferating glial cells to vacillate in their selection of a specific lineage. This situation demonstrates developmental PLASTICITY and the ability of glia to adapt to an altered environment. Whether committed glial cells in situ can be induced to switch their lineage when normal CNS conditions are altered is an intriguing question that remains to be answered.

Observations of hamster sperm‐egg fusion in freeze‐fracture replicas including the use of filipin as a sterol marker

We have extended the observations of previous transmission electron microscopy studies of sperm-egg fusion to include those of freeze-fracture replicas showing sperm-egg interactions before, during, and following sperm head fusion with the egg membrane. Hamster eggs were incubated with hamster sperm under polyspermic conditions and were observed after a period of 5-30 minutes. After fixation, the eggs and sperm were exposed to filipin, which binds beta-OH-sterols to form visible complexes in freeze-fracture replicas. Filipin can act as a marker for egg plasma membrane wherein it is abundant, while filipin is relatively scarce in the acrosome-reacted hamster sperm membrane, found only in the plasma membrane of the equatorial segment. The earliest sperm-egg interactions are observed between the egg microvilli and the perforatorium and the equatorial segment of the sperm, and the initial fusion between egg and sperm occurs in the vicinity of the equatorial segment. At later stages of fusion involving the postacrosomal segment, a clear line of demarcation is observed between the filipin-rich egg membrane and the filipin-poor sperm postacrosomal segment, suggesting that filipin binding lipids from the egg intercalate into the sperm membrane following membrane fusion. The anterior segment of the sperm does not fuse with the egg but is instead incorporated into a cytoplasmic vesicle derived from both sperm and egg membranes. In this latter step, filipin-sterol complexes are not found in sperm-derived membranes suggesting that there may be barriers to the movement of filipin binding lipids from the egg into these sperm membranes.

Ultrastructural localization of immunoglobulin M in activated B-cells in patients with epidemic hemorrhagic fever using gold probe

Our previous electron microscopic studies have shown for the first time that the atypical lymphoid cells in the peripheral blood of patients with epidemic hemorrhagic fever (EHF) during the acute febrile phase of the illness appeared to be activated B-cells and plasma cells of various developmental stages. But it is still not a tall clear whether those initially activated cells were actually B-cells. In this study, an indirect immuno gold staining method for the detection of B-cell surface antigen was used.Peripheral blood mononuclear cells were isolated from anticoagulated venous blood of 3 EHF patients (2—7 days of illness) and control donors by centrifugation at 2,500 Xg for 15 min. The cells obtained from the buffy coat were washed in TBS and resuspended in TBS-5% BSA. Unfixed cells were in cubated insuspension in step I with monoclonal antibody (MS27) directed against human IgM (μ Chain) for 30 min at 37±C. After washing in TBS—BSA, the gold probe was applied to the cells in step II incubation with GAM-G15 reagent for 45 min at RT. The gold probe was prepared in our laboratory according to Slot and Geuze. After the third washing in TBS-BSA, the cell pellets were fixed, dehydrated and embedded in Epon 812.

Computer-Aided Three-Dimensional Images of the Helical Structure in the Apical Tubule of Absorbing Epithelia

Computer-aided three-dimensional models of the helical structure within an apical tubule (AT) of several absorbing epithelia (kidney proximal tubule, visceral yolk sac, and ductuli efferentes) were constructed using ray-tracing graphics software to further understand the highly ordered structural configuration. Our previous electron microscopic studies using thin-section technique have first revealed the helical structure within the AT fixed in situ with a mixture of formaldehyde, glutaraldehyde, and osmium tetroxide. In the present study, we construct a computer-aided three-dimensional model of the helical structure in the AT to explain quantitative data obtained by the electron microscopy. The model could well explain several aspects of electron microscopical images and enables us to understand more clearly the three-dimensional configuration of the unique structure associated with the AT.

HREM study of M, Y and W hexagonal type ferrites

Hexagonal ferrites have similar structural symmetries and are built by the same structural units. HREM has proved to be an excellent tool to identify individual stacking bloks, in M, Y and W hexaferrites.M, Y and W hexagonal type ferrites are well-known magnetic materials for applications. Some previous electron microscopy studies on these ferrites, reveal a variety of new phases coming from the microsyntactic intergrowth of M, Y and W structures. We report in this paper some microstructural features of these ferrites.BaFe12O19 (M) hexagonal ferrite was synthetized by using the standard ceramic technique. Crystals of Y type ferrite (nominal composition: BaLi0.5Fe0.5ZnFe16027) were prepared from stoichiometric amounts of BaCO3, ZnO and LiCO3. The synthesis of W-type ferrite (formula: Ba2Zn2Fe16027) is described elsewhere.Samples for observation were prepared by grinding powders of the above ferrites under acetone. HREM study was performed at the "Centro de Microscopía Electrónica, Univ. Complutense" using both 400 Kv and 200 Kv electron microscopes (JEM-400EX and JEM-200FX)

A new concept of fibrin formation based upon the linear growth of interlacing and branching polymers and molecular alignment into interlocked single-stranded segments.

In a previous electron microscopic study of early fibrin polymers processed by freeze drying and rotatory shadowing, a large proportion of loosely constructed, frequently branching linear molecular chains was observed; their structural organization was inconsistent with a half-staggered double-stranded model for fibrin polymerization. These conflicting results prompted us to investigate the structure of early fibrin polymers prepared according to a large variety of methods currently used for electron microscopy of macromolecules. By use of a systematic random sampling procedure, fibrin polymers were photographically recorded. They were classified according to their morphological form, and the frequency of occurrence of each configuration was determined. Half-staggered double-stranded forms accounted for less than 1% of all types encountered. Interpretation of the structural organization manifested in the diverse polymer forms observed necessitated the construction of a new interlocked single-strand model for fibrin polymerization. The fibrin polymerization process combines simultaneous propagation of linear growth, branching, and lateral interlocking (leading to lateral association), resulting in the rapid formation of a fibrin network. The structural pattern developing during growth of fibrin polymers appears to be determined principally by the enzymatic mechanism and not solely by the intrinsic molecular structure of fibrinogen. The validity of the interlocked single-strand model was tested by selective fibrinopeptide-B-releasing experiments. Under such activation conditions, the polymer forms predicted according to this and the half-staggered double-strand models should differ; the structures observed were indeed consistent with the interlocked single-strand hypothesis. The compatibility of existing data with this model is discussed.

Focal points for chromosome condensation and decondensation revealed by three-dimensional in vivo time-lapse microscopy

Although the dynamic behaviour of chromosomes has been extensively studied in their condensed state during mitosis, chromosome behaviour during the transition to and from interphase has not been well documented. Previous electron microscopic studies suggest that chromosomes condense in a non-uniform fashion at the nuclear periphery. But chromosome condensation is a complicated and dynamic process and requires continuous observation in living tissues to be fully understood. Using a recently developed three-dimensional time-lapse fluorescence microscopy technique, we have observed chromosomes as they relax from telophase, through interphase, until their condensation at the next prophase. This technique has been improved to produce higher-resolution images by implementing new stereographic projection and computational processing protocols. These studies have revealed that chromosomal regions on the nuclear envelope, distinct from the centromeres and telomeres, serve as foci for the decondensation and condensation of diploid chromosomes. The relative positions of the late decondensation sites at the beginning of interphase appear to correspond to the early condensation sites at the subsequent prophase.

Posttranslational insertion of a membrane protein on Caenorhabditis elegans sperm occurs without de novo protein synthesis.

We have examined the mechanism of membrane protein insertion in the ameboid spermatozoa of Caenorhabditis elegans using two monoclonal antibodies which recognize the same set of eight sperm-specific polypeptides. Previous electron microscopic studies demonstrated that these antibodies label surface and cytoplasmic populations of antigen. Cells whose surface antigen had been removed by proteolysis were able to localize new membrane protein insertion at the tips of pseudopodial projections. C. elegans sperm do not contain the protein synthesizing machinery needed for delivery of new membrane to the cell surface. It has, therefore, been of interest to determine how localized membrane assembly occurs. Here we have determined the subcellular location of each of these eight polypeptides. A closely positioned doublet of bands around 97 kD (comprising 40% of the total antigen in sperm) represents surface (larger member of doublet) and cytoplasmic (lower member) forms of protein. Proteolysis of live cells eliminated this surface form from immunoblots but did not affect the cytoplasmic protein. When cells were allowed to reinsert new protein following removal of the enzyme, this surface form was regenerated. Since sperm are unable to synthesize new protein, this higher molecular weight species may arise from a posttranslational modification of proteins in the cytoplasmic pool. We present evidence suggesting that the surface protein is generated from this cytoplasmic pool by addition of fatty acid. Fatty acid acylation would account for both the observed decrease in electrophoretic mobility of the surface form and provide increased hydrophobicity to the protein which may allow for its insertion into the lipid bilayer.