Cell differentiation in embryogenesis involves extensive changes in gene expression structural reorganization within the nucleus, including chromatin condensation and nucleoprotein immobilization. We hypothesized that nuclei in naive stem cells would therefore prove to be physically plastic and also more pliable than nuclei in differentiated cells. Micromanipulation methods indeed show that nuclei in human embryonic stem cells are highly deformable and stiffen 6-fold through terminal differentiation, and that nuclei in human adult stem cells possess an intermediate stiffness and deform irreversibly. Because the nucleo-skeletal component Lamin A/C is not expressed in either type of stem cell, we knocked down Lamin A/C in human epithelial cells and measured a deformability similar to that of adult hematopoietic stem cells. Rheologically, lamin-deficient states prove to be the most fluidlike, especially within the first Ϸ10 sec of deformation. Nuclear distortions that persist longer than this are irreversible, and fluorescence-imaged microdeformation with photobleaching confirms that chromatin indeed flows, distends, and reorganizes while the lamina stretches. The rheological character of the nucleus is thus set largely by nucleoplasm/chromatin, whereas the extent of deformation is modulated by the lamina. chromatin remodeling ͉ cell mechanics ͉ nuclear plasticity

Nuclear lamins comprise the nuclear lamina, a scaffoldlike structure that lines the inner nuclear membrane. B-type lamins are present in almost all cell types, but A-type lamins are expressed predominantly in differentiated cells, suggesting a role in maintenance of the differentiated state. Previous studies have shown that lamin A/C is not expressed during mouse development before day 9, nor in undifferentiated mouse embryonic carcinoma cells. To further investigate the role of lamins in cell phenotype maintenance and differentiation, we examined lamin expression in undifferentiated mouse and human embryonic stem (ES) cells. Wide-field and confocal immunofluorescence microscopy and semiquantitative reverse transcription-polymerase chain reaction analysis revealed that undifferentiated mouse and human ES cells express lamins B1 and B2 but not lamin A/C. Mouse ES cells display high levels of lamins B1 and B2 localized both at the nuclear periphery and throughout the nucleoplasm, but in human ES cells, B1 and B2 expression is dimmer and localized primarily at the nuclear periphery. Lamin A/C expression is activated during human ES cell differentiation before downregulation of the pluripotency marker Oct-3/4 but not before the downregulation of the pluripotency markers Tra-1-60, Tra-1-81, and SSEA-4. Our results identify the absence of A-type lamin expression as a novel marker for undifferentiated ES cells and further support a role for nuclear lamins in cell maintenance and differentiation.

The study of cell locomotion is fundamental to such diverse processes as embryonic development, wound healing and metastasis. Since microtubules play a role in establishing the leading lamellum and maintaining cell polarity, it is important to understand their dynamic behaviour. In vitro, subunits exchange with polymer by treadmilling and by dynamic instability. Disassembly events can be complete (catastrophic) or incomplete (tempered). In vivo, microtubules are in dynamic equilibrium with subunits with a half-time for turnover of 4-20 min. Microtubules grow by elongation of their ends and are replaced one by one with turnover being most rapid at the periphery. Although previous results are consistent with dynamic instability, we sought to directly test the mechanism of turnover. Direct observations of fluorescent microtubules in the fibroblast lamellum show that individual microtubules undergo rounds of assembly and disassembly from the same end. Reorganization of the microtubule network occurs by a tempered mode of dynamic instability.

Abstract. During the movement of chromosomes in anaphase, microtubules that extend between the kinetochores and the poles shorten. We sought to determine where subunits are lost from these microtubules during their shortening. Prophase or prometaphase cells on coverslips were injected with fluoresceinated tubulin and allowed to progress through mitosis. Immediately after the onset of anaphase, a bar-shaped beam of laser light was used to mark a domain on the kinetochore fibers by photobleaching a band, ",,1.0 Ixm wide, across the spindle. In different cells, spindles were photobleached at varying distances from the chromosomes. Cells were allowed to continue in anaphase until the chromosomes had further separated. They were then lysed, fixed, and prepared for double-label immunofluorescence with an antibody to fluorescein that does not bind appreciably to bleached fluorescein, and with an antibody to tubulin. Photobleached domains of microtubules appeared as bands of reduced fluorescence in the anti-fluorescein image. However, the anti-tubulin labeling revealed that microtubules were present and continuous through the photobleached domains. In all cases, the chromosomes approached and invaded the bleached domain while the bleached domain itself remained stationary with respect to the near pole. These results demonstrate that the chromosomes move along stationary kinetochore microtubules and that depolymerization of these microtubules during anaphase takes place at the kinetochore. In contrast to the generally accepted older view that chromosomes are passive objects pulled by "traction fibers; we suggest that the kinetochore is an active participant in generating the motive force that propels the chromosome to the pole.