To study the dynamics of elastic fiber assembly, mammalian cells were transfected with a cDNA construct encoding bovine tropoelastin in frame with the Timer reporter. Timer is a derivative of the DsRed fluorescent protein that changes from green to red over time and, hence, can be used to distinguish new from old elastin. Using dynamic imaging microscopy, we found that the first step in elastic fiber formation is the appearance of small cell surface-associated elastin globules that increased in size with time (microassembly). The elastin globules are eventually transferred to pre-existing elastic fibers in the extracellular matrix where they coalesce into larger structures (macroassembly). Mechanical forces associated with cell movement help shape the forming, extracellular elastic fiber network. Time-lapse imaging combined with the use of Timer constructs provides unique tools for studying the temporal and spatial aspects of extracellular matrix formation by live cells.

Elastic fibers are responsible for the extensibility and resilience of many vertebrate tissues, and improperly assembled elastic fibers are implicated in a number of human diseases. It was recently demonstrated that in vitro, cells first secrete tropoelastin into a punctate pattern of globules. To study the dynamics of macroassembly, that is, the assembly of the secreted tropoelastin globules into elastic fibers, we utilized long-term time-lapse immunofluorescence imaging and a tropoelastin p Timer fusion protein, which shifts its fluorescence spectrum over time. Pulse-chase immunolabeling of the fibroblast-like RFL-6 cells demonstrates that tropoelastin globules aggregate in a hierarchical manner, creating progressively larger fibrillar structures. By analyzing the correlation between cell and extracellular matrix movements, we show that both the aggregation process and shaping the aggregates into fibrillar form is coupled to cell motion. We also show that the motion of non-adjacent cells becomes more coordinated as the physical size of elastin-containing aggregates increases. Our data imply that the formation of elastic fibers involves the concerted action and motility of multiple cells.

High grade gliomas are characterized by bad prognosis and poor response to therapy. The unexpected motogenic effect of low-dose radiation and paclitaxel treatments highlight the importance of similar investigations to develop more effective clinical treatments.

A novel assay based on micropatterning and time-lapse microscopy has been developed for the study of nuclear migration dynamics in cultured mammalian cells. When cultured on 10-20-microm wide adhesive stripes, the motility of C6 glioma and primary mouse fibroblast cells is diminished. Nevertheless, nuclei perform an unexpected auto-reverse motion: when a migrating nucleus approaches the leading edge, it decelerates, changes the direction of motion, and accelerates to move toward the other end of the elongated cell. During this process, cells show signs of polarization closely following the direction of nuclear movement. The observed nuclear movement requires a functioning microtubular system, as revealed by experiments disrupting the main cytoskeletal components with specific drugs. On the basis of our results, we argue that auto-reverse nuclear migration is due to forces determined by the interplay of microtubule dynamics and the changing position of the microtubule organizing center as the nucleus reaches the leading edge. Our assay recapitulates specific features of nuclear migration (cell polarization, oscillatory nuclear movement), while it allows the systematic study of a large number of individual cells. In particular, our experiments yielded the first direct evidence of reversive nuclear motion in mammalian cells, induced by attachment constraints.