CYTOARCHITECTURE OF SIZE-EXCLUDING COMPARTMENTS IN LIVING CELLS

By fluorescence ratio imaging of large and small inert tracer particle s in living cells, we have previously shown that particles 24 nm in ra dius are excluded from otherwise uncharacterized compartments in the d istal and perinuclear cytoplasm (Luby-Phelps, K. and Taylor, D.L., 198 8. Cell Motil. Cytoskel. 10, 28-37). In this study we examined the cyt oarchitecture of these compartments. Whole-mount TEM showed that dista l size-excluding compartments were devoid of membrane-bounded organell es and were filled with a dense cytomatrix consisting of numerous, lon g bundles of thin filaments interconnected by a more random meshwork o f short thin filaments. The mean diameter of void spaces in the cytoma trix of distal excluding compartments was 31 nm, compared to 53 nm in adjacent nonexcluding domains. The height of the distal excluding comp artments was generally less-than-or-equal-to 50% of the height in the adjacent non-excluding compartment. An electron-dense structure having the same projected outline as the perinuclear size-excluding compartm ent was visible by whole-mount TEM, but the cells were too thick and o smiophilic in this region to resolve any detail. Immunofluorescence lo calization of cytoskeletal proteins in distal excluding compartments i ndicated the presence of filament bundles containing F-actin, nonmuscl e filamin (ABP280) and alpha-actinin. F-actin and ABP280, but not alph a-actinin, were found also in between these filament bundles. Microtub ules and vimentin generally were rare or absent from distal excluding domains. Staining of living cells with DMB-ceramide revealed that the perinuclear size-excluding compartment consisted of a compact, juxtanu clear domain coinciding with the trans-Golgi, surrounded by a more dif fuse domain coinciding with a perinuclear concentration of endoplasmic reticulum. Intense immunofluorescence staining for vimentin was also observed in the perinuclear size-excluding compartment. We propose tha t the most likely mechanism for exclusion from distal compartments is molecular sieving by a meshwork of actin filament bundles interconnect ed by an F-actin/ABP280 gel network, while exclusion from the perinucl ear compartment may be due to close apposition of cisternae in the tra ns-Golgi and a network or basket of vimentin filaments in the centroso mal region of the cell.