Our studies suggest that Nudel and dynein function upstream of LB3 to promote assembly of LB3-containing spindle matrix in a MT-dependent manner. We show that Nudel is required for transiently concentrating LB3 onto MTs, probably through dynein-mediated transport of LB3 toward the minus ends of MTs. This could promote both LB3 self-association and association of LB3 with other components of the spindle matrix. Very little is known about the polymerization state of lamin and its mechanism of regulation in the interphase nuclear lamina or in the mitotic spindle. The finding that Nudel interacts with lamin B should help to further study the mechanism of lamin B assembly in mitosis.
Both Nudel and dynein have been found to participate in the organization of vimentin 10
and neurofilaments 2
in the interphase cytoplasm of tissue culture cells and in neurons, respectively. In neurons, Nudel interacts directly with NF-L. Here, we show that Nudel interacts with yet another intermediate filament, lamin B. Importantly, we demonstrate that Nudel directly interacts with the region of lamin B that is conserved among all intermediate filament proteins. By interacting with both the intermediate filaments and MTs, Nudel and dynein may have a general role in coordinating the functions of the two cytoskeletal structures in animal cells.
Dynein functions in spindle pole focusing in animal cells 18, 20, 21
. Our findings suggest that the dynein pathway regulates spindle pole organization, in part, by facilitating the assembly of lamin B into the spindle matrix in a MT dependent manner. Dynein is also known to facilitate the transport of NuMA to minus ends of MTs where NuMA helps to focus the minus ends into spindle poles 21
. We have shown previously that NuMA localizes to the lamin-containing spindle matrix 8
. Therefore, the dynein pathway may regulate spindle matrix assembly by targeting multiple components of the matrix, including lamin B and NuMA, to orchestrate spindle organization.
Nudel and dynein also have a well-established role in regulating membrane trafficking along MTs. Since we have shown that lamin B spindle matrix contains membranes and is disrupted by detergent, it is tempting to speculate that Nudel and dynein may use MTs to concentrate and incorporate multiple spindle matrix components, including lamin B, NuMA, and certain membrane compartments, into a functional spindle matrix. Such type of matrix may not only help MTs to regulate chromosome segregation but also use MTs to regulate the partitioning of cell fate determinants and membrane systems during cell division. Consistent with this, membrane-associated enzymes have been shown to regulate spindle morphogenesis 9, 22–24
. Moreover, our mass-spectrometry analyses of the isolated lamin B spindle matrix have identified both SAFs and proteins with known functions in membrane trafficking and in cell fate determination (Table S1
Conceptually, it is interesting to consider that the spindle matrix might organize the cell during mitosis in a similar manner as the interphase nucleus (Fig. S10
). The interphase nuclear lamina helps to organize the nuclear structure and function through its interactions with transcription and replication machineries 25, 26
. By binding to the nuclear envelope proteins, such as Man1 and nesprins, the nuclear lamina also mediates the connection between the nucleus and the cytoplasmic membrane systems as well as the cytoskeleton 27
. Although the nuclear envelope and lamina undergo disassembly in mitosis, it is possible that many components of the disassembled nucleus become organized into the mitotic spindle matrix to facilitate the organization of the membrane system and nuclear content for cell division.
Interestingly, in lower eukaryotes that undergo ‘closed’ mitosis with the spindle assembles inside the intact nuclear envelope, dynein is required for nuclear movement and mitotic spindle and or nucleus orientation, but not for spindle assembly per se 28, 29
. The lack of an obvious requirement for dynein in ‘closed’ mitosis could be because that the intact mitotic nucleus functions as the spindle matrix to organize both the nuclear and cytoplasmic contents for division (Fig. S10
). Our studies suggest that one important function of dynein and Nudel in animal cell mitosis where the nuclear envelope breaks down (‘open’ mitosis) is to help organize a membranous lamin B matrix that resembles the nuclear envelope and lamina.