A novel PKC phosphorylation site in lamin B3 influences both nuclear lamina dynamics and nuclear size in Xenopus laevis embryos and extracts. PKC activity also modulates nuclear size in mammalian cell culture, and a conserved phosphorylation site in human lamin A affects the association of lamin A with the nuclear envelope and nuclear size.

The ability to visualize cytoskeletal proteins and their dynamics in living cells has been critically important in advancing our understanding of numerous cellular processes, including actin- and microtubule-dependent phenomena such as cell motility, cell division, and mitosis. Here we describe a novel set of fluorescent protein fusions designed specifically to visualize microtubules in living systems using fluorescence microscopy. Each fusion contains a fluorescent protein module linked in frame to a modified phospho-deficient version of the microtubule-binding domain of Tau (mTMBD). We found that expressed and purified constructs containing a single mTMBD decorated Xenopus egg extract spindles more homogenously than similar constructs containing the microtubule-binding domain of Ensconsin, suggesting that the binding affinity of mTMBD is minimally affected by localized signaling gradients generated during mitosis. Furthermore, microtubule dynamics were not grossly perturbed by the presence of Tau-based fluorescent protein fusions. Interestingly, the addition of a second mTMBD to the opposite terminus of our construct caused dramatic changes to the spatial localization of probes within spindles. These results support the use of Tau-based fluorescent protein fusions as minimally perturbing tools to accurately visualize microtubules in living systems.

Striking size variations are prominent throughout biology, at the organismal, cellular, and subcellular levels. Important fundamental questions concern organelle size regulation and how organelle size is regulated relative to cell size, also known as scaling. Uncovering mechanisms of organelle size regulation will inform the functional significance of size as well as the implications of misregulated size, for instance in the case of nuclear enlargement in cancer. Xenopus egg and embryo extracts are powerful cell-free systems that have been utilized extensively for mechanistic and functional studies of various organelles and subcellular structures. The open biochemical nature of the extract permits facile manipulation of its composition, and in recent years extract approaches have illuminated mechanisms of organelle size regulation. This review largely focuses on in vitro Xenopus studies that have identified regulators of nuclear and spindle size. We also discuss potential relationships between size scaling of the nucleus and spindle, size regulation of other subcellular structures, and extract experiments that have clarified developmental timing mechanisms. We conclude by offering some future prospects, notably the integration of Xenopus extract with microfluidic technology.

Emerin is an inner nuclear membrane protein often mutated in Emery–Dreifuss muscular dystrophy. Because emerin has diverse roles in nuclear mechanics, cytoskeletal organization, and gene expression, it has been difficult to elucidate its contribution to nuclear structure and disease pathology. In this study, we investigated emerin’s impact on nuclei assembled in Xenopus laevis egg extract, a simplified biochemical system that lacks potentially confounding cellular factors and activities. Notably, these extracts are transcriptionally inert and lack endogenous emerin and filamentous actin. Strikingly, emerin caused rupture of egg extract nuclei, dependent on the application of shear force. In egg extract, emerin localized to nonnuclear cytoplasmic membranes, and nuclear rupture was rescued by targeting emerin to the nucleus, disrupting its membrane association, or assembling nuclei with lamin A. Furthermore, emerin induced breakage of nuclei in early-stage X. laevis embryo extracts, and embryos microinjected with emerin were inviable, with ruptured nuclei. We propose that cytoplasmic membrane localization of emerin leads to rupture of nuclei that are more sensitive to mechanical perturbation, findings that may be relevant to early development and certain laminopathies.