We investigated how nuclear size is regulated in two related but different sized frog species as well as during early frog development, two physiological examples of nuclear scaling. Using Xenopus
egg extracts to examine intrinsic mechanisms of nuclear scaling in the absence of the cell showed that titratable cytoplasmic factors regulate nuclear size to a greater extent than DNA content and that differences in the concentrations of importin α and Ntf2 are sufficient to explain most of the observed interspecies nuclear scaling by altering nuclear import. Importin α, but not Ntf2, also plays a role in nuclear scaling during embryogenesis in X. laevis
. While nucleocytoplasmic transport was known to be required for NE growth (D’Angelo et al., 2006
; Neumann and Nurse, 2007
; Newport et al., 1990
), our data show that titrating nuclear import concomitantly scales nuclear size, and that this mechanism can account for how the size of the nucleus is controlled in two frog species and during development.
Importin α mediates nuclear scaling by regulating overall import of NLS cargos, consistent with computer modeling and cell culture experiments showing that importin α concentration positively correlates with the rate and steady-state level of nuclear import (Görlich et al., 2003
; Riddick and Macara, 2005
; Smith et al., 2002
). However, our results indicate a more complex relationship between nuclear import factors and nuclear size. For example, we observe that increasing importin α concentration more than 3-fold over normal levels reduces nuclear size (, S3B
) probably because elevated lamin B3 import that occurs under these conditions (data not shown) is detrimental to nuclear assembly (Figure S4A
). Ntf2 has also been implicated as a positive regulator of both Ran and bulk import (Riddick and Macara, 2005
). While the Ntf2-Ran relationship holds true in our experiments, we find that increased Ntf2 slows import of large cargos, such as Qdots, but not smaller proteins like GFP-NLS. Since it associates with the NPC, Ntf2 could influence import rates based on cargo size (Clarkson et al., 1996
). In fact, studies of X. laevis
oogenesis revealed that late stage oocytes acquire the ability to import large nucleoplasmin-coated gold particles concomitantly with a reduction in Ntf2 levels (Feldherr et al., 1998
). Furthermore, addition of Ntf2 to those oocytes reduced import of gold particles, similar to our observation that increasing the Ntf2 concentration in X. laevis
reduced Qdot import (). It is worth noting that supplementing X. laevis
extract with Ntf2 up to the X. tropicalis
level slowed but did not block Qdot import, suggesting other interspecies NPC differences may affect cargo size-dependent import.
Nuclear size appears to be determined by import of specific NLS cargos, not by mass action transport. LB3 was a good candidate since its import is importin α-mediated, it is required for NE expansion (Jenkins et al., 1993
; Newport et al., 1990
), and its overexpression induces proliferation of nuclear membrane (Goldberg et al., 2008
; Prufert et al., 2004
). Addition of LB3, but not Npl or GFP-NLS, to X. tropicalis
egg extract increased nuclear size, but not to the size of X. laevis
, suggesting additional NLS proteins are involved. Potential nuclear sizing cargos include inner nuclear membrane proteins that interact with the lamina, like the lamin B receptor and LAPs, as well as SUN and KASH family proteins that span the NE and mediate interactions between the nucleus and cytoskeleton. Consistent with this idea, NPC manipulations that increase translocation of integral membrane proteins to the inner NE correlate with increased nuclear size (Theerthagiri et al., 2010
). The fact that Qdot import positively correlates with nuclear size indicates that cargos important for scaling are relatively large. Although lamin monomers are only 70kD, they minimally form tetramers made up of two dimers, each composed of 50 nm elongated coiled-coils (Aebi et al., 1986
; Heitlinger et al., 1991
). Since particles as large as 20-megadaltons can transit the X. laevis
NPC, LB3 may be imported as large oligomers.
We discovered some striking similarities between interspecies nuclear size regulation and nuclear scaling during Xenopus embryogenesis. Reductions in nuclear size during development were accompanied by diminishing import capacity for NLS cargos, and scaling of nuclear size at the MBT correlated with a drop in total and nuclear importin α levels. Increasing the concentration of importin α in embryos increased nuclear size without noticeably affecting development, suggesting that nuclear size per se does not regulate early developmental transitions. Thus, conserved importin α-mediated transport mechanisms regulate nuclear size both during development and between frog species, but distinct and yet uncharacterized mechanisms also contribute to nuclear scaling in Xenopus embryogenesis.
Our data suggest two nuclear sizing regimes determined by either reaction rates or abundance of NE components. The egg is stockpiled in order to form approximately 4000 MBT nuclei, and therefore these components are not limiting in egg extracts and early embryos. In this regime, nuclear size is determined by rates of NE expansion and nuclear import in conjunction with cell cycle timing. In contrast, MBT nuclei reach a steady-state size when import and NE components like lamins are no longer in excess. Consistent with this idea, increasing importin α expression in MBT embryos caused nuclei to reach a new steady-state size () where lamins became limiting since co-expressing importin α and LB3 further augmented nuclear size (data not shown). Interestingly, the amount of LB3 loaded into the eggs of each species correlates well with the total NE surface area at the MBT, with X. laevis
containing 2.1-fold more total LB3 than X. tropicalis
at the onset of development and 2-fold more NE at the MBT when transcription starts (Table S1
). Since the ratio of NE surface area to embryo volume at this transition is 2.1-fold higher in the smaller X. tropicalis
species (Table S1
), the starting LB3 concentration in the egg is also about 2-fold higher (). Thus Xenopus
eggs are loaded with the proper amount of LB3, and presumably other nuclear envelop components, so that they are not limiting during the rapid divisions of early development.
Our results are consistent with multiple, mutually non-exclusive models of organelle size control. Considering a static model, importin α and Ntf2 levels limit nuclear import of LB3, thereby constraining the rate at which nuclei expand. However, dynamic processes must balance import-mediated growth. Nuclear size is a regulated cellular parameter that depends on tissue type, developmental state as demonstrated during Xenopus
embryogenesis, and species as shown comparing X. laevis
and X. tropicalis
, in which nuclear size differences have evolved by fine-tuning the expression of nuclear import factors. A fundamental question is why nuclear size is regulated. Changes in the dimensions and morphology of the nucleus are associated with pathologies including cancer (Webster et al., 2009
; Zink et al., 2004
), but dissecting the cause and effect relationship between nuclear size and disease state is difficult. Understanding the role that nuclear import plays in scaling nuclear size and identifying relevant factors and their mechanisms of action provide an avenue to directly manipulate nuclear size in the context of normal and diseased cells in order to examine the functional consequences.