Keratin filaments restrict organelle migration into the forming spindle of newt pneumocytes

Abstract

When viewed by light microscopy the mitotic spindle of newt pneumocytes appears to assemble in an optically clear area of cytoplasm, virtually devoid of mitochondria and other organelles, which is often much larger than the spindle. This clear area is also frequently larger than the region previously occupied by the nucleus. It forms even in prometaphase cells depleted of microtubules prior to nuclear envelope breakdown by colchicine treatment. Time-lapse video microscopy reveals that as prometaphase proceeds this clear area slowly and progressively collapses around the forming spindle so that it is greatly diminished or nonexistent by the onset of anaphase. The sharply defined nature of the boundary between the clear area and the remaining cytoplasm and the fact that organelles accumulate at its periphery suggest that a structural barrier is present at the boundary that limits organelle migration into the forming spindle. Immunofluorescence and electron microscopy, of cells previously followed in the living state, reveal that the periphery of the clear area contains prominent bundles of keratin filaments but lacks microtubules and actin. From our observations we conclude that keratin filaments form a loosely organized cage that surrounds the forming newt pneumocyte spindle. We propose that this cage functions, in part, to restrict the dispersion of chromosomes during nuclear envelope breakdown and to impede the bulk migration of organelles into the forming spindle.