Norman Lieska

Intermediate Filaments: Possible Functions as Cytoskeletal Connecting Links Between the Nucleus and the Cell Surface

Intermediate filaments (IF), along with microtubules and microfilaments constitute the three major fibrous protein systems that have been defined by electron microscopical, biochemical, and immunological studies of a wide variety of cell types.’-’ In addition to the major proteinaceous subunits making up the backbone or core of these cytoplasmic fibers (e.g. tubulin in microtubules), there are also numerous “associated proteins” which appear to complex with their walls. These associated proteins are thought to be involved in various functions, including crosslinking and the regulation of polymerization. Numerous examples of such associated proteins can be found in the literature including many of the actin or microfilament associated proteins such as tropomyosin, a-actinin, filamin, etc.,’” and the microtubule-associated proteins (MAPS), both of which have been described extensively. Recently, IF-associated proteins (IFAPs) have also been described and activity in this area of study will undoubtedly increase in intensity in the near future.’.’ When considered together these three fiber systems and their associated proteins constitute a major proportion of total cell protein which collectively has become known as the cytoskeleton or the cytoskeletal system. Unfortunately, the term “cytoskeleton” when used in this fashion, is a misleading one, as it implies a lack of dynamic activity and further suggests that these three distinctive fibrous protein systems are similar with regard to their functions. This is certainly not the case and is contrary to well established facts that demonstrate that the three cytoplasmic fiber systems are distinctly different with regard to their subcellular organization, their relationship to different cell functions, their relative stabilities, and their biochemical and immunological properties.’-’ In spite of these considerations, numerous recent biochemical and molecular studies have emphasized associations of various cellular components (for example, nuclei, ribosomes, etc.) with nondescript “cytoskeletal elements” that are usually

Immunotyping of radial glia and their glial derivatives during development of the rat spinal cord

SummaryThe differentiation of glia in the central nervous system is not well understood. A major problem is the absence of an objective identification system for involved cells, particularly the early-appearing radial glia. The intermediate filament structural proteins vimentin and glial fibrillary acidic protein have been used to define the early and late stages, respectively, of astrocyte development. However, because of the non-specificity of vimentin and the temporal overlap in expression patterns of both proteins, it is difficult to refine our view of the process. This is especially true of the early differentiation events involving radial glia. Using the developmentally-expressed intermediate filament-associated protein IFAP-70/280 kD in conjunction with vimentin and glial fibrillary acidic protein markers, a comprehensive investigation of this problem was undertaken using immunofluorescence microscopy of developing rat spinal cord (E13-P28 plus adult). The phenotypes of the cells were defined on the basis of their immunologic composition with respect to IFAP-70/280 kD (I), vimentin (V) and GFAP (G). A definitive immunotype for radial glia was established, viz, I+/V+/G−; thus reliance upon strictly morphological criteria for this early developmental cell was no longer necessary. Based upon the immunotypes of the cells involved, four major stages of macroglial development were delineated: (1) radial glia (I+/V+/G−); (2) macroglial progenitors (I+/V+/G+); (3) immature macroglia (I−/V+/G+); and (4) mature astrocytes (I−/V+/G+ primarily in white matter and I−/V−/G+, the predominant type in gray matter). It is of interest to note that the cells of the floor plate were distinguished from radial glia by their lack of IFAP-70/280 kD immunoreactivity. Introduction of the IFAP-70/280 kD marker has therefore provided a more refined interpretation of the various differentiation stages from radial glia to mature astrocytes.

A subpopulation of reactive astrocytes at the immediate site of cerebral cortical injury

We have identified an early-appearing intermediate filament-associated protein (IFAP-70/280 kDa) in radial glia and their immediate derivatives. This IFAP is absent in the adult CNS. In this study, we examined the reexpression of this early glial differentiation trait in rat reactive astrocytes induced by stab injury of the cerebrum. Double-label immunofluorescence microscopy demonstrated that by 36 h postlesion, IFAP-70/280 kDa was present in a few GFAP-positive astrocytes in the area adjacent to the wound. As the gliotic reaction progressed, the number of IFAP-positive reactive astrocytes increased and by 5-6 days postlesion, IFAP-70/280 kDa was present in most of the hypertrophied astrocytes in tissue immediately adjacent to the wound. By 8 days postlesion, while the number of IFAP-negative reactive astrocytes away from the wound diminished, the IFAP-containing reactive astrocytes close to the wound persisted. Concurrently, they began to change from a stellate form to an elongated shape, with their longitudinal axes radiating from the wound. The immunoreactivity of this IFAP started to diminish at 20 days postlesion, and by 30 days postlesion, it was not observed in the remaining gliotic cells. These results demonstrate that reactive astrocytes induced by stab-wound injury can be divided into two subtypes: persistent IFAP-70/280 kDa-containing cells which are close to the wound in the area of the glial scar and transient IFAP-70/280 kDa-negative cells which are farther from the wound. The reappearance of IFAP-70/280 kDa also suggests that some reactive astrocytes have the capacity to recapitulate early developmental stages.

Proteins of the intermediate filament cytoskeleton as markers for astrocytes and human astrocytomas

There is a pressing need for a more accurate system of classifying human astrocytomas, one that is based on morphologic characteristics and that could also make use of distinctive biochemical markers. However, little is known about the phenotypic characteristics of astrocytomas. Recent studies have shown that the expression of proteins comprising the intermediate filament (IF) cytoskeleton of astrocytic cells is developmentally regulated. It is our hypothesis that this changing protein profile can be used as the basis of a system for clearly and objectively classifying astrocytomas. A spectrum of human astrocytomas has been examined by immunofluorescence microscopy employing antibodies to several IF structural subunit proteins (GFAP, vimentin, and keratins) and an IF-associated protein IFAP-300kDa. These proteins occupy unique temporal niches in the cytogenesis of the astrocytic cells: keratins in cells of the neuroectoderm; vimentin and IFAP-300kDa in radial glia and immature glia; GFAP in mature astrocytes; and vimentin in some mature astrocytes. In agreement with previous reports, our immunofluorescence studies have revealed both GFAP and vimentin in all astrocytoma specimens. Two new observations, however, are of particular interest: IFAP-300kDa is detectable in all astrocytic tumors, and the proportion of keratin-containing cells present in the astrocytomas is in direct relationship to the degree of the malignancy. Because IFAP-300kDa is not present in either normal mature or reactive astrocytes, this protein appears to represent a specific marker of transformed (malignant) astrocytes. If it is presumed that higher malignancy grades represent the most dedifferentiated cellular state of the astrocytes, the presence of keratin-containing cells is not totally unexpected, given the ectodermal (epithelial) origin of the CNS. Specific developmentally regulated proteins of the IF cytoskeleton thus appear to hold great potential as diagnostic markers of astrocytomas and as tools for investigating the biology of these tumors.

Expression and distribution of cytoskeletal IFAP-300kD as an index of lens cell differentiation.

By their implication in the organization of the intermediate filament (IF) cytoskeleton, IF-associated proteins (IFAPs) can delineate subsets of the same IF type within a cell; moreover, they are proving useful as markers of the differentiation states of certain cells. For these reasons the expression of the vimentin-associated IFAP-300kD was investigated in the constantly differentiating cell lineage of the adult bovine lens. Immunofluorescence microscopy and immunoblot analysis were employed using a monoclonal anti-IFAP-300kD and a rabbit anti-lens vimentin. Cultures of adult lens epithelial cells were immunopositive for the IFAP. By double-label studies the IFAP-300kD pattern co-localized with that of the vimentin-type IF; moreover, the IFAP pattern co-distributed with that of both colchicine-sensitive and -insensitive IF systems. IFAP-300kD was also present in a co-distributing pattern with vimentin IF in fresh lens epithelial cells on whole mounts. There was a differential expression of the IFAP in the lens fiber cells in that those of the cortex exhibited the IFAP and vimentin IF, while both proteins were absent from the nuclear fiber cells. Furthermore, there was a differential distribution of the IFAP within the cortical fiber cells in that the IFAP localized only with a paramembranal subset of IF. Immunoblot analysis supported the presence of IFAP-300kD in the lens cytoskeletal fraction. IFAP-300kD thus identified a subset of vimentin IF whose location may have functional significance for the cortical fiber cell. The changes in the IFAPs expression and distribution pattern throughout lens cell differentiation in the adult organ suggest the usefulness of IFAP-300kD as a potential marker in studying lens cell differentiation in vitro.

Immunofluorescence and biochemical studies of the type VI collagen expression by human glioblastoma cells in vitro

The human glioblastoma cell line U-87 MG was found to express a 140 kD polypeptide which was recognized on immunoblot analysis by a monoclonal antibody to type VI collagen. This polypeptide was digestible by a highly purified bacterial collagenase. After treatment of U-87 MG cells by pepsin, the protein profile revealed the two major pepsin-resistant fragments identical in Mr to those of collagen VI extracted from human placenta. The respective peptide maps from V8 protease one-dimensional gels of these two fragments were identical to those obtained with human collagen VI. Immunofluorescent staining by antibodies to type VI collagen was observed in the extracellular matrix. Moreover, U-87 MG cells were found to be positive for A2B5, a cell surface marker specific for O-2A type glial precursor cells. These data indicate that the human glioblastoma cell line U-87 MG exhibits the properties of glial precursor cells and expresses collagen type VI in vitro. This cell line therefore may prove valuable for comparative investigations of the regulation of type VI collagen synthesis, and may be useful as a model to study the function and pathological importance of type VI collagen in human brain tumours, both in vitro and in vivo.

Distinct developmental subtypes of cultured non-stellate rat astrocytes distinguished by a new glial intermediate filament-associated protein

The nature and tissue origin of cultured non-stellate astrocytes have not been defined. On the basis of immunofluorescence microscopy using multiple double-labeling with antibodies to glial fibrillary acidic protein (GFAP), vimentin, and the recently identified intermediate filament-associated protein (IFAP)-70/280kD, four distinct astrocytic subtypes were definable in neonatal rat brain astrocytes in culture. All of these were of the non-stellate type on the basis of morphology. Similar examination of developing rat cerebral cortex identified these same subtypes as distinct differentiation states of astrocytes. These findings indicate that the parallel developmental events can be studied in vitro.

Immunomicroscopy of neurofilaments in chromaffin cells of the adult bovine adrenal gland.

Neurofilaments (NFs) represent a class of intermediate filaments which are highly specific for neurons. The most abundant of the native NFs is the 68 kD subunit (NF‐L). Chromaffin cells of the adrenal medulla express NF subunits under culture conditions. However, NF expression in situ is questionable. It has been reported that as chromaffin cell precursors mature and begin to express catecholamine‐synthesizing enzymes, their neuronal traits are extinguished and they become endocrine‐like cells. This study reports that while NF‐L may be lacking in the adrenal medulla of some species, NF‐L immunoreactivity is clearly present in the adult bovine adrenal medulla. Immunofluorescence microscopy of bovine chromaffin cells in culture demonstrated NF immunoreactivity localized to both thin, highly ramified filaments present throughout the cell and frequently to an intensely immunofluorescent spot located near the nucleus. Double‐label immunofluorescence microscopy and immunoblot analysis also demonstrated NF‐L immunoreactivity in mature chromaffin cells of the bovine adrenal gland. In vitro and in situ immunofluorescence results were confirmed by immunogold and immunoperoxidase labelling, respectively. In both cases, NF‐L immunoreactivity was associated with filaments in close proximity to the nucleus. Additionally, a spheroidal aggregate of immunogold‐labelled NFs was seen adjacent to the nucleus in cultured cells. In conclusion, NF‐L in bovine chromaffin cells demonstrates that at least one neuronal trait persists in these catecholamine‐producing cells of the mature adrenal gland. In addition, this study emphasizes the fact that interspecies comparisons must be interpreted with caution, especially when attempting to formulate a generalized hypothesis.