Structural proteins in the growth cone of cultured spinal cord neurons

The distribution of several structural proteins in the extending neurites and growth cones of cultured embryonic mouse spinal cord neurons was studied by indirect immunofluorescence, using affinity chromatography-purified antibodies. Fibroblastic cells in the same cultures served as internal standards for the evaluation of staining intensities. Anti-tubulin, anti-actin, and anti-clathrin stained neurons and their processes intensely, while staining with anti-α-actinin was only moderate compared with fibroblasts. Microtubules were resolved by anti-tubulin in the ‘palm’ of the growth cone but not in the neurite. Anti-actin stained even the finest lamellae and filopodia of the growth cone, and the neurite. Anti-α-actinin revealed an irregularly speckled pattern of cross-reactive material in the neurite and in the palm of the growth cone and was absent from the filopodia. Anti-clathrin stained the neurite intensely and homogeneously, and to a lesser extent the palm of the growth cone. The staining with antibodies against tubulin and clathrin differed grossly between neurons and fibroblastic cells. Within the neuron there were only gradual differences in staining intensities. The growth cone was not qualitatively different from the rest of the neurite, except for the filopodia which lacked tubulin and α-actinin, similar to the microvilli of epithelial cells.     

Trypanosoma cruzi: distribution of fluorescently labeled tubulin and actin in epimastigotes

Cytoskeletal components were visualized in epimastigote forms of Trypanosoma cruzi by double immunofluorescence microscopy using monospecific antibodies against tubulin and against actin. Intense staining of the flagellum and the edges of the cell body was observed when the cells were stained with anti-tubulin, reflecting the presence of the basal bodies, the flagellar axoneme and the subpellicular microtubules. A less intense staining was seen in the cell body of epimastigotes stained with anti-actin. However, an intense staining was observed with this antibody in the flagellum, in a pattern similar to that observed with anti-tubulin. It is suggested that the paraxial structure, which is formed by a complex array of 6-nm-thick microfilaments is composed, at least in part, of actin.     

Comparison of purified anti-actin and fluorescent-heavy meromyosin staining patterns in dividing cells

We purified actin antibodies by affinity chromatography from the serum of rabbits immunized with glutaraldehyde-fixed chicken gizzard actin filaments and used this anti-actin to localize actin in myofibrils and fixed cultured cells at each stage of the cell cycle. By double immunodiffusion the anti-actin reacted with both smooth and skeletal muscle actin. Several blocking and absorption experiments demonstrated that the antibodies also bound specifically to actin in nonmuscle cells. The same structures stained using either the direct or the indirect fluorescent antibody technique; and, while the indirect method was more sensitive, the direct method was superior because there was no detectable nonspecific staining. As expected, anti-actin stained the I-band of myofibrils. It also stained stress fibers and membrane ruffles in HeLa cells. Some PtK-2 cells have straight stress fibers which stained with anti-actin, but in confluent cultures all PtK-2 cells have, instead, sinuous phase-dense fibers which stained with antibody. At prophase the whole cytoplasm stained uniformly with anti-actin. During metaphase and anaphase, anti-actin staining was concentrated diffusely in the mitotic spindle. In contrast, fluorescent heavy meromyosin stained discrete fine spindle fibers in these fixed cells. During cytokinesis, anti-actin stained the whole cytoplasm uniformly and was not concentrated in the cleavage furrow.     

Effects of cytochalasin B on fibroblasts,lymphoid cells,and platelets revealed by human anti-actin antibodies

Human antibodies against actin revealed on smeared lymphoblastoid cells a strong staining of numerous microvilli of different lengths extending from the cell surface. Smeared human platelets stained by anti-actin serum showed a bright cytoplasmic fluorescence and projections extending from the surface. Human fibroblasts spread on glass were multi-shaped, and anti-actin serum revealed brightly stained fibers running through the cells. After treatment with cytochalasin B, all types of cells investigated became rounded up, and surface projections could not be demonstrated. The staining pattern indicated a redistribution of the cellular contractile proteins after cytochalasin B treatment. Cytochalasin B did not impair the antigenicity of actin, since presence of the drug did not influence the antibody absorbing capacity of actin.Culture of lymphoblastoid cells and human fibroblasts in the presence of colchicin did not influence the staining pattern of actin antibodies.     

Distribution of microtubules during the breakdown of the nuclear envelope of the Xenopus oocyte: an immunocytochemical study

Xenopus oocytes were stained by anti-tubulin and anti-MAP1 antibodies during the first meiotic cell division. In the prophase-blocked oocytes, only few microtubules are present around the upper part of the nuclear envelope. These microtubules are resistant to cold, calcium and antimitotic drug treatments. At this stage, monoclonal anti-MAP1 antibody and polyclonal anti-centrosome antibody reveal punctate staining of the nucleus and nucleoli. During the progesterone-induced maturation, a microtubular network appears at the basal part of the disrupting nucleus. Anti-MAP1 and anti-centrosome antibodies stain a dense layer at the basal part of this microtubular array. Microtubules present in this array are cold, calcium- and antimitotic drug sensitive. Anti-MAP1 and anti-tubulin antibodies stain the whole metaphase II spindle, whereas only the poles of the metaphase II spindle are stained by the anti-centrosome antibody.     

Unusual organization of desmin intermediate filaments in muscular dysgenesis and TTX-treated myotubes

Cytoskeletal intermediate filaments were studied in muscular dysgenesis (mdg) and tetrodotoxin-treated inactive mouse embryo muscle cultures during myofibrillogenesis. Both muscular dysgenesis and tetrodotoxin-treated muscles are characterized in vitro by a total lack of contractile activity and an abnormal development of myofibrils. We studied the organization of the microtubule and intermediate filament networks with immunofluorescence, using anti-tubulin, anti-vimentin, and anti-desmin antibodies during normal and mdg/mdg myogenesis in vitro. Mdg/mdg myotubes present a heterogeneous microtubule network with scattered areas of decreased microtubule density. At the myoblast stage, cells expressed both vimentin and desmin. After fusion only desmin expression is revealed. In mutant myotubes the desmin network remains in a diffuse position and does not reorganize itself transversely, as it does during normal myogenesis. The absence of a mature organization of the desmin network in mdg/mdg myotubes is accompanied by a lack of organization of myofibrils. The role of muscle activity in the organization of myofibrils and desmin filaments was tested in two ways: (i) mdg/mdg myotubes were rendered active by coculturing with normal spinal cord cells, and (ii) normal myotubes were treated with tetrodotoxin (TTX) to suppress contractions. Mdg/mdg innervated myotubes showed cross-striated myofibrils, whereas desmin filaments remained diffuse. TTX-treated myotubes possessed disorganized myofibrils and a very unusual pattern of distribution of desmin: intensively stained desmin aggregates were superimposed upon the diffuse network. We conclude, on the basis of these results, that myofibrillar organization does not directly involve intermediate filaments but does need contractile activity.     

Distinction between smooth muscle,fibroblasts and endothelial cells in culture by the use of fluoresceinated antibodies against smooth muscle actin

Summary FITC-labelled antibodies against native actin from chicken gizzard smooth muscle (Gröschel-Stewart et al., 1976) have been used to stain cultures of guinea-pig vas deferens and taenia coli, rabbit thoracic aorta, rat ventricle and chick skeletal muscle. The I-band of myofibrils of cardiac muscle cells and skeletal muscle myotubes stains intensely. In isolated smooth muscle cells, the staining is located exclusively on long, straight, non-interrupted fibrils which almost fill the cell. Smooth muscle cells which have undergone morphological dedifferentiation to resemble fibroblasts with both phase-contrast microscopy and electronmicroscopy still stain intensely with the actin antibody. In those muscle cultures which contain some fibroblasts or endothelial cells, the non-muscle cells are not stained with the actin antibody even when the reactions are carried out at 37° C for 1 h or after glycerination. Prefusion skeletal muscle myoblasts also do not stain with this antibody.It is concluded that the actin antibody described in this report is directed against a particular sequence of amino acids in muscle actin which is not homologous with non-muscle actin. The usefulness of this antibody in determining the origin of cells in certain pathological conditions such as atherosclerosis is discussed.This work was supported by the Life Insurance Medical Research Fund of Australia and New Zealand, the National Heart Foundation of Australia, the Deutsche Forschungsgemeinschaft and the Wellcome Trust (London). We thank Janet D. McConnell for excellent technical assistance     

Microtubule-associated protein 2 within axons of spinal motor neurons: associations with microtubules and neurofilaments in normal and beta,beta'-iminodipropionitrile-treated axons

We have examined the distribution of microtubule-associated protein 2 (MAP2) in the lumbar segment of spinal cord, ventral and dorsal roots, and dorsal root ganglia of control and beta,beta'-iminodipropionitrile- treated rats. The peroxidase-antiperoxidase technique was used for light and electron microscopic immunohistochemical studies with two monoclonal antibodies directed against different epitopes of Chinese hamster brain MAP2, designated AP9 and AP13. MAP2 immunoreactivity was present in axons of spinal motor neurons, but was not detected in axons of white matter tracts of spinal cord and in the majority of axons of the dorsal root. A gradient of staining intensity among dendrites, cell bodies, and axons of spinal motor neurons was present, with dendrites staining most intensely and axons the least. While dendrites and cell bodies of all neurons in the spinal cord were intensely positive, neurons of the dorsal root ganglia were variably stained. The axons of labeled dorsal root ganglion cells were intensely labeled up to their bifurcation; beyond this point, while only occasional central processes in dorsal roots were weakly stained, the majority of peripheral processes in spinal nerves were positive. beta,beta'- Iminodipropionitrile produced segregation of microtubules and membranous organelles from neurofilaments in the peripheral nervous system portion and accumulation of neurofilaments in the central nervous system portion of spinal motor axons. While both anti-MAP2 hybridoma antibodies co-localized with microtubules in the central nervous system portion, only one co-localized with microtubules in the peripheral nervous system portion of spinal motor axons, while the other antibody co-localized with neurofilaments and did not stain the central region of the axon which contained microtubules. These findings suggest that (a) MAP2 is present in axons of spinal motor neurons, albeit in a lower concentration or in a different form than is present in dendrites, and (b) the MAP2 in axons interacts with both microtubules and neurofilaments.     

A specific effect of muscle cells on the distribution of presynaptic proteins in neurites and its absence in a C2 muscle cell variant

The distribution of neurofilament (NF) and synaptic vesicle (SV) proteins in neurites cultured in vitro was visualized with immunocytochemical methods. NF and SV proteins were detected in neurites from both embryonic mouse spinal cord and chick ciliary ganglion neurons. NF proteins generally occupied more proximal, unbranched neurite segments while SV proteins were most often found in highly branched terminal segments. Neurites from mouse spinal cord cells showed a striking segregation of the NF and SV proteins into distinct domains; neurites from chick ciliary ganglion cells exhibited a similar, though less pronounced segregation. In cocultures of neurons and muscle cells, the neurite segments in contact with myotubes more often stained for SV than for NF while the opposite was true for neurites not in contact with myotubes. The preferential association of SV neurites with myotubes was also observed when the myotubes were previously fixed with paraformaldehyde. This association was absent in neurites growing over Chinese hamster ovary cells, suggesting that the effect is specific for muscle cells. Coculture of neurons with variant strains of C2 myotubes that are deficient in AChR (1R-) or proteoglycans (S27) revealed a preferential association of SV neurites with 1R- myotubes but not with S27 myotubes. Thus, proteoglycans on the surface of C2 myotubes may influence the growth and/or differentiation of presynaptic neurons.     

Relationship of primary and secondary myogenesis to fiber type development in embryonic chick muscle.

The formation of fast and slow myotubes was investigated in embryonic chick muscle during primary and secondary myogenesis by immunocytochemistry for myosin heavy chain and Ca2(+)-ATPase. When antibodies to fast or slow isoforms of these two molecules were used to visualize myotubes in the posterior iliotibialis and iliofibularis muscles, one of the isoforms was observed in all primary and secondary myotubes until very late in development. In the case of myosin, the fast antibody stained virtually all myotubes until after stage 40, when fast myosin expression was lost in the slow myotubes of the iliofibularis. In the case of Ca2(+)-ATPase, the slow antibody also stained all myotubes until after stage 40, when staining was lost in secondary myotubes and in the fast primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis. In contrast, the antibodies against slow muscle myosin heavy chain and fast muscle Ca2(+)-ATPase stained mutually exclusive populations of myotubes at all developmental stages investigated. During primary myogenesis, fast Ca2(+)-ATPase staining was restricted to the primary myotubes of the posterior iliotibialis and the fast region of the iliofibularis, whereas slow myosin heavy chain staining was confined to all of the primary myotubes of the slow region of the iliofibularis. During secondary myogenesis, the fast Ca2(+)-ATPase antibody stained nearly all secondary myotubes, while primaries in the slow region of the iliofibularis remained negative. Thus, in the slow region of the iliofibularis muscle, these two antibodies could be used in combination to distinguish primary and secondary myotubes. EM analysis of staining with the fast Ca2(+)-ATPase antibody confirmed that it recognizes only secondary myotubes in this region. This study establishes that antibodies to slow myosin heavy chain and fast Ca2(+)-ATPase are suitable markers for selective labeling of primary and secondary myotubes in the iliofibularis; these markers are used in the following article to describe and quantify the effects that chronic blockade of neuromuscular activity or denervation has on these populations of myotubes.     

Immunohistochemical observations on tubulin and alpha actinin during mitosis of cultured fibroblasts

In this work we studied alpha-actinin and tubulin sites in rabbit fibroblasts in culture. Antibodies anti-alpha-actinin were used for indirect PAP-reaction while antibodies anti-tubulin were used for indirect immunofluorescence method. The observations were carried out by light microscopy, phase-contrast and interference-contrast microscopy, with regard to actinin, and by fluorescence microscopy, with regard to tubulin. During the early mitotic phase, alpha-actinin is localized all over the cell membrane of the fibroblasts, forming a sort strong protective cap, while during diacinesis it forms only rings, localizing below the cell membrane and the philopodia. Thus tubulin forms the bundle fibres during mitotic phases.     

Alpha-actinin localization in the cleavage furrow during cytokinesis

We used antibodies against alpha-actinin and myosin labeled directly with contrasting fluorochromes to localize these contractile proteins simultaneously in dividing chick embryo cells. During mitosis anti-alpha-actinin stains diffusely the entire cytoplasm including the mitotic spindle, while in the same cells intense antimyosin staining delineates the spindle. During cytokinesis both antibodies stain the cleavage furrow intensely, and until the midbody forms the two staining patterns in the same cell are identical at the resolution of the light microscope. Thereafter the anti-alpha-actinin staining of the furrow remains strong, but the antimyosin staining diminishes. These observations suggest that alpha-actinin participates along with actin and myosin in the membrane movements associated with cytokinesis.     

Localization of myosin IIB at the leading edge of growth cones from rat dorsal root ganglionic cells.

Primary cultures of rat dorsal root ganglionic (DRG) cells were stained with isoform-specific antibodies against non-muscle myosin II. Antibodies against the brain type myosin (MIIB) stained the peripheries of growth cones and non-neuronal cells. Double staining of the cells with the anti-myosin antibodies and rhodamine-phalloidin or anti-actin antibodies indicated that MIIB co-exists, with F-actin, at the leading edge. Antibodies against platelet myosin stained neither leading edges nor neurites, but stained the cell bodies of neurons and the stress fibers of non-neuronal cells. These results suggest that MIIB functions in the motility of the leading edge of DRG cells.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai Virus) M protein

Summary Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Monoclonal antibody to rat brain actin antigenically enhanced with HVJ (Sendai virus) M protein

Monoclonal antibody to rat brain actin was easily produced using HVJ (Sendai Virus) M protein to enhance the antigenicity of the actin. This monoclonal antibody was determined to be IgM with a kappa light chain. By immunoblot analysis the antibody was also shown to react with rat brain actin but not with HVJ M protein on nitrocellulose sheets. Utilizing the antibody, neuronal cytoplasm in the cerebral cortex, the anterior and posterior horns in the spinal cord, the spinal ganglion and astrocytes showed positive immunohistochemical staining by light microscopy. However, Purkinje cells showed variable staining, some staining intensely, while others were negative. All of neurons in specific anatomical locations showed always positive staining but variable intensities. Vascular walls were stained only faintly. By electron microscopy, neuronal cytoplasm showed diffuse positive staining. Other areas showed a positive reaction, including dendrites, the postsynaptic densities, and a few capillary endothelial cells and arterial smooth muscle cells. The results suggest that the HVJ M protein was effective for producing monoclonal antibody to brain actin, and that the antibody could be utilized for the immunohistochemical study of neuronal elements in both normal and pathological conditions.     

Widespread distribution of the major polypeptide component of MAP 1 (microtubule-associated protein 1) in the nervous system

We prepared a monoclonal antibody to microtubule-associated protein 1 (MAP 1), one of the two major high molecular weight MAP found in microtubules isolated from brain tissue. We found that MAP 1 can be resolved by SDS PAGE into three electrophoretic bands, which we have designated MAP 1A, MAP 1B, and MAP 1C in order of increasing electrophoretic mobility. Our antibody recognized exclusively MAP 1A, the most abundant and largest MAP 1 polypeptide. To determine the distribution of MAP 1A in nervous system tissues and cells, we examined tissue sections from rat brain and spinal cord, as well as primary cultures of newborn rat brain by immunofluorescence microscopy. Anti-MAP 1A stained white matter and gray matter regions, while a polyclonal anti-MAP 2 antibody previously prepared in this laboratory stained only gray matter. This confirmed our earlier biochemical results, which indicated that MAP 1 is more uniformly distributed in brain tissue than MAP 2 (Vallee, R.B., 1982, J. Cell Biol., 92:435-442). To determine the identity of cells and cellular processes immunoreactive with anti-MAP 1A, we examined a variety of brain and spinal cord regions. Fibrous staining of white matter by anti-MAP 1A was generally observed. This was due in part to immunoreactivity of axons, as judged by examination of axonal fiber tracts in the cerebral cortex and of large myelinated axons in the spinal cord and in spinal nerve roots. Cells with the morphology of oligodendrocytes were brightly labeled in white matter. Intense staining of Purkinje cell dendrites in the cerebellar cortex and of the apical dendrites of pyramidal cells in the cerebral cortex was observed. By double-labeling with antibodies to MAP 1A and MAP 2, the presence of both MAP in identical dendrites and neuronal perikarya was found. In primary brain cell cultures anti-MAP 2 stained predominantly cells of neuronal morphology. In contrast, anti-MAP 1A stained nearly all cells. Included among these were neurons, oligodendrocytes and astrocytes as determined by double-labeling with anti-MAP 1A in combination with antibody to MAP 2, myelin basic protein or glial fibrillary acidic protein, respectively. These results indicate that in contrast to MAP 2, which is specifically enriched in dendrites and perikarya of neurons, MAP 1A is widely distributed in the nervous system.     

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.     

Immunological analysis of chick notochord and cartilage matrix development with antisera to cartilage matrix macromolecules

Transverse frozen sections from the postcephalic region of stage 9-16 chick embryos and from the wing bud region of stage 17-31 embryos were stained with antibodies to the major extracellular matrix components of cartilage. These probes included unfractionated A1 and A2 antisera to the major cartilage proteoglycan, affinity-purified purified antibodies to the proteoglycan core protein and to Type II collagen, and a monoclonal antibody to keratan sulfate. In embryos as early as stage 10, notochord stained specifically with the keratan sulfate monoclonal antibody. At this stage the notochord, as well as surrounding tissues, were negative to cartilage proteoglycan and collagen antibodies. Positive staining with the latter probes was coordinately acquired by notochord cells and their accompanying sheath around stage 15, while surrounding tissues remained negative. At this stage, the ventral region of the perispinal cord sheath exhibited light staining with the proteoglycan and keratan sulfate antibodies though failing to react to Type II collagen antibodies. Positive staining of notochord and ventral spinal cord persisted through later developmental stages. As revealed by immunofluorescence, definitive vertebral chondroblasts first emerged at approximately stage 23 and definitive limb chondroblasts at stage 25. The results are discussed in terms of the possible multiple roles of notochord in early embryogenesis.     

Fibroblasts promote the formation of a continuous basal lamina during myogenesis in vitro

Analyses were made of the requirements for the formation of a continuous basal lamina during myogenesis of quail muscle in vitro. A culture system was developed in which mass cultures of differentiating muscle cells were embedded in a native gel of rat tail collagen. Fibroblastic cells, which were also present in the cultures, migrated into the gel and within a few days surrounded the newly formed myotubes. In this environment, a continuous basal lamina was formed at the surface of the myotubes as demonstrated by immunofluorescent staining with monoclonal antibodies against type IV collagen, laminin, and heparan sulfate, as well as by electron microscopic immunolocalization. To distinguish between the role of the fibroblasts and the collagen gel in promoting basal lamina formation, clones of quail muscle cells lacking fibroblasts were subsequently embedded in a native rat tail collagen gel. Under these conditions, only very limited fluorescent staining for basement membrane components was observed associated with the myotubes. However, the introduction of chick muscle or skin fibroblasts into the clonal cultures just before gel formation resulted in the formation of an extensive basal lamina on the surface of the myotubes. Conditioned medium from fibroblast cultures by itself was not effective in promoting basal lamina formation. These results clearly show that during myogenesis in vitro fibroblasts must be in close proximity to the myotubes for a continuous basal lamina to form. These results probably relate closely to the interactions that must occur during myogenesis in vivo between the muscle cells and the surrounding connective tissue including the developing tendons.