The nuclear lamina is a fundamental constituent of metazoan nuclei. It is composed mainly of lamins, which are intermediate filament proteins that assemble into a filamentous meshwork, bridging the nuclear envelope and chromatin 1–4. Besides providing structural stability to the nucleus 5,6, the lamina is involved in many nuclear activities, including chromatin organization, transcription and replication 7–10. However, the structural organization of the nuclear lamina is poorly understood. Here, we use cryo-electron tomography (cryo-ET) to obtain a detailed view of the organization of the lamin meshwork within the lamina. Data analysis of individual lamin filaments resolves a globular-decorated fiber appearance and shows that A- and B-type lamins assemble into tetrameric 3.5 nm thick filaments. Thus, lamins exhibit a structure that is remarkably different from the other canonical cytoskeletal elements. Our findings define the architecture of the nuclear lamin meshworks at molecular resolution, providing insights into their role in scaffolding the nuclear lamina.

The KinI kinesin MCAK is a microtubule depolymerase important for governing spindle microtubule dynamics during chromosome segregation. The dynamic nature of spindle assembly and chromosome-microtubule interactions suggest that mechanisms must exist that modulate the activity of MCAK, both spatially and temporally. In Xenopus extracts, MCAK associates with and is stimulated by the inner centromere protein ICIS. The inner centromere kinase Aurora B also interacts with ICIS and MCAK raising the possibility that Aurora B may regulate MCAK activity as well. Herein, we demonstrate that recombinant Aurora B-INCENP inhibits Xenopus MCAK activity in vitro in a phosphorylationdependent manner. Substituting endogenous MCAK in Xenopus extracts with the alanine mutant XMCAK-4A, which is resistant to inhibition by Aurora B-INCENP, led to assembly of mono-astral and monopolar structures instead of bipolar spindles. The size of these structures and extent of tubulin polymerization in XMCAK-4A extracts indicate that XM-CAK-4A is not defective for microtubule dynamics regulation throughout the cytoplasm. We further demonstrate that the ability of XMCAK-4A to localize to inner centromeres is abolished. Our results show that MCAK regulation of cytoplasmic and spindle-associated microtubules can be differentiated by Aurora B-dependent phosphorylation, and they further demonstrate that this regulation is required for bipolar meiotic spindle assembly. INTRODUCTIONAssembly of the microtubule-based bipolar spindle, the apparatus that powers chromosome segregation during mitosis and meiosis (M phase), is driven by the inherent dynamic instability of microtubules, as well as by proteins that alter microtubule dynamics (Hyman and Karsenti, 1996;Wittmann et al., 2001). Accordingly, M-phase onset triggers global, cytoplasmic changes in microtubule dynamics that expedite spindle assembly, most notably an increase in the frequency of catastrophes, the transition from microtubule growth to shrinkage (Belmont et al., 1990;Verde et al., 1992;Rusan et al., 2001;Kinoshita et al., 2002). In Xenopus egg extracts, the KinI kinesin MCAK, classified by its internally located motor domain (Wordeman and Mitchison, 1995;Walczak et al., 1996), is essential for governing this change in catastrophe frequency (Walczak et al., 1996;Tournebize et al., 2000). Biochemical studies indicate that MCAK likely accomplishes catastrophe induction directly by catalytically depolymerizing microtubules from their protofilament ends (Desai et al., 1999b;Hunter et al., 2003).Although spindle assembly demands global regulation of microtubule dynamics, proper spindle function also requires precise control of microtubule dynamics regulation at defined locations within the spindle. Because accurate chromosome segregation requires that each kinetochore of a pair of sister chromatids be attached to microtubules derived from opposing spindle poles (biorientation), it is particularly important to control the interaction of spindle microtubules with kinetochores (Rieder and Salmon, 1...

Molecular interactions are the basic language of biological processes. They establish the forces interacting between the building blocks of proteins and other macromolecules, thus determining their functional roles. Because molecular interactions trigger virtually every biological process, approaches to decipher their language are needed. Single-molecule force spectroscopy (SMFS) has been used to detect and characterize different types of molecular interactions that occur between and within native membrane proteins. The first experiments detected and localized molecular interactions that stabilized membrane proteins, including how these interactions were established during folding of alpha-helical secondary structure elements into the native protein and how they changed with oligomerization, temperature, and mutations. SMFS also enables investigators to detect and locate molecular interactions established during ligand and inhibitor binding. These exciting applications provide opportunities for studying the molecular forces of life. Further developments will elucidate the origins of molecular interactions encoded in their lifetimes, interaction ranges, interplay, and dynamics characteristic of biological systems.

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