Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.

Abstract

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization . We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis . First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer . The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely 'unaltered by detergent extraction . Residual desmosomes, as can be seen in thin sections, were also well-preserved . The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation . The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders . This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts . Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold . Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders . The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions . The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold .A potentially powerful addition to the study of cell structure is afforded by combining detergent extraction with unembedded whole mount electron microscopy. In this protocol, soluble proteins are extracted with non-ionic detergent under near physiological conditions of ionic strength and pH. The cellular structure that remains after detergent extraction is called the skeletal framework (1, 2). The elaborate filament framework can be clearly seen using whole mount transmission electron microscopy, which omits conventional embedding, sectioning, and staining. We have observed the three-

Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

“…We used CSK buffer (C) with Triton (C+ T) as the basic conditions, which have been used earlier to extract majority of the cellular proteins without destroying the cellular architecture and permit visualization of NER and other repair proteins recruited to the damaged DNA [15][16][17][18] . To this buffer, we added 0.42 M NaCl (C+ T+ S), since we had earlier seen that 0.42 M NaCl retained chromatin-bound PARP-1 during cell fractionation in vitro 7 whereas 1.6 M NaCl was shown to strip almost all PARP-1 from cells 19 .…”

Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nuclear structure

“…This model is in direct contrast to many current models of cell mechanics, which envision the viscous fluid-like cytoplasm and surrounding elastic membrane to be the major load-bearing elements in living cells (13)(14)(15). On the other hand, microscopic studies demonstrate structural continuity between ECM molecules, transmembrane proteins, CSK filaments, and nuclear scaffolds in detergent-extracted cells (16)(17)(18). However, the mechanical relevance of these structural interconnections remains unclear.…”

“…On the other hand, microscopic studies demonstrate structural continuity between ECM molecules, transmembrane proteins, CSK filaments, and nuclear scaffolds in detergent-extracted cells (16)(17)(18). However, the mechanical relevance of these structural interconnections remains unclear.…”

Tight Junction Proteins Form Large Complexes and Associate with the Cytoskeleton in an ATP Depletion Model for Reversible Junction Assembly

“…Since detergent extractability is an established biochemical means for analyzing protein-cytoskeletal interactions (8,18,19,31), we examined the Triton X-100 solubility properties of TJ proteins (ZO-1, ZO-2, and cingulin) after metabolic inhibitor treatment. As shown in Fig.…”