Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

Abstract

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.The finding that replication of fibroblasts in culture is restricted to a defined time span introduces the establishment of a methodology for in vitro observation of cellular senescence. Not only have the initial reports of Swim and Parker (26) and Hayflick and Moorehead (8) been repeatedly confirmed in fibroblasts different from the original WI-38, but also this may be observed in cells of nonmesenchymal origin, such as endothelial and smooth muscle cells (see review in reference 20). In contrast to Hayflick's interpretation that loss of proliferative potential by human diploid fibroblasts in culture is a manifestation of aging, Martin et al. (15) and Bell et al. (1) suggest that the cessation of replication can be interpreted as the final event in differentiation.An increase in cell size and the presence of large rigid filamentous structures are two of the frequently observed phenotypic properties associated with the process of fibroblast senescence (3,28,33). Our recent results demonstrate that the filamentous structures commonly seen in the large, nonproliferating senescent cells are composed of numerous actincontaining microfilaments, intermediate filaments (IFs) 1, and an elaborate network of microtubules (29). These cytoskeletal components form a complex three-dimensional structure that occupies most of the cytoplasm.In apparently normal fibroblasts of a proliferating monolayer culture, individual IFs are in general in...

Absence of three secreted proteins and presence of a 57-kDa protein related to irreversible arrest of cell growth.

“…Human fibroblasts display heterogeneity in cell size and replicative activity as they proliferate in culture. In early stages of their in vitro lifespan (CPDL < 10), cultures of the GM0011 and GM3529 strains are comprised of a fairly homogeneous population of rapidly dividing, small, and spindle-shaped cells (18,19). In the GM0011 strain, the cell population remains relatively homogeneous until the CPDL reaches 42.…”

“…4, lane 1). Note that a very short period (CPDL = [19][20][21][22] exists in the in vitro lifespan of GM3529, immediately before the onset of cellular senescence, during which the cells stop secreting the 80-, 84-, and 87-kDa proteins but have not yet begun secreting the 57-kDa protein (Fig. 4, lane 2).…”

“…From such studies (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), the control of cell proliferation appears to involve a number ofcellular proteins, the expression ofwhich is correlated with the proliferation state of a cell. Transition into the S phase of the cell cycle is accompanied by induction of some of these proteins (such as cyclin) and concurrent repression of another (such as statin (14,18,19). Human fibroblasts display heterogeneity in cell size and replicative activity as they proliferate in culture.…”

“…[3H]thymidine incorporation (14,19). Fibroblasts at the early and late stages of their in vitro lifespan described above will be referred to as young and senescent, respectively.…”

“…All cultures were grown and maintained in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (vol/vol) and 1% nonessential amino acids at 370C in a humidified atmosphere of 5% CO2 as described (14,18,19 England Nuclear; 1 Ci = 37 GBq) in MEM containing 0.5% fetal bovine serum and unlabeled L-methionine (0.8 pug/ml) at 370C for the indicated times. After labeling, culture media (conditioned media) were collected and centrifuged at 500 x g for 10 min at 40C.…”