Samwer M et al. (2013), The nuclear F-actin interactome of Xenopus oocy...

XB-ART-47205

EMBO J 2013 Jul 03;3213:1886-902. doi: 10.1038/emboj.2013.108.

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The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis.

Samwer M, Dehne HJ, Spira F, Kollmar M, Gerlich DW, Urlaub H, Görlich D.

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Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.

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Species referenced: Xenopus laevis
Genes referenced: actl6a aicda gnl3 kif14 kif14l npm1 ptk2 tbx2

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	Figure 1. Compartment-specific F-actin interactomes of Xenopus oocytes. (A) Scheme of the phalloidin-based affinity matrix. Phalloidin is shown in blue, the PEG–silane-based linker in green, the solid phase in black and the passivating PEG coat in orange. The control matrix contains a methoxy-PEG group instead of phalloidin. See Supplementary Figure S1 for more details. (B) Scheme for preparing clean nuclear and cytoplasmic oocyte extracts. Oocytes were punctured with forceps at the animal pole and nuclei were isolated into aqueous buffer by application of mild pressure to the oocyte's equatorial plane. (C) F-actin complexes isolated from nuclear and cytoplasmic extracts, analysed by SDS–PAGE/Coomassie staining. See Table I for complete list of identified nuclear F-actin interactors. (D) Nuclear and cytoplasmic fractions were immunoblotted with antibodies raised against identified nuclear F-actin interactors. Tubulin, eEF1A and Arp2 served as cytoplasmic markers; nucleoplasmin is a nuclear marker. Since the loads had been normalised to the compartments' volumes (nucleus ∼50 nl, yolk-free cytoplasm ∼500 nl), the blots directly compare nuclear and cytoplasmic concentrations. (E) Immunoblot analysis of proteins bound from a nuclear extract to phalloidin beads. NabKin and Supervillin bound strongly to the matrix; binding of nucleoplasmin was undetectable. (F) Immunoblot analysis of NabKin expression in various oocyte and embryo developmental stages. eEF1A served as loading control.
	Figure 2. NTEs of NabKin and KIF14 bundle F-actin. (A) A simplified neighbour-joining tree of the kinesins-3 family, including the paralogues NabKin and KIF14. (B) Predicted domain organisation of NabKin and KIF14 (Xl, Xenopus laevis; Xt, Xenopus tropicalis; Hs, Homo sapiens). In contrast to other kinesin-3 family members, NabKin and the KIF14s possess unusually long NTEs. The NabKin and KIF14–NTEs share no obvious sequence similarity (indicated by distinct colouring). (C) Differential centrifugation assay was used for distinguishing actin-bundling and actin-binding factors from non-interacting proteins. Candidate proteins (3 μM) were mixed with actin (10 μM) in F-actin buffer and tested for mutual interaction as outlined in the scheme on the left. Fractions obtained were analysed by SDS–PAGE and Coomassie staining. Red asterisks mark the F-actin-bundling NTEs and the green asterisk marks Drebrin, which binds, but does not bundle F-actin (Sekino et al, 2007). The image was cut at black lines and rearranged for clarity. The NabKin–NTE had been fused to an N-terminal His14-SUMO-tag in the experiments shown in 2C+D; KIF14–NTEs (XtKIF14aa1-341 and Hs KIF14aa1-345) were untagged. (D) Electron micrographs of negatively stained F-actin filaments obtained in the absence or presence of indicated NTEs. Scale bars=200 nm. (E) NabKin colocalises with nuclear F-actin in Xenopus oocyte cryostat sections. Analysis was by confocal laser-scanning microscopy (CLSM). Dashed line marks the nuclear envelope, separating nuclear (N) and cytoplasmic (Cy) compartment. The F-actin-free intranuclear regions probably represent the nucleoli. The white square marks the area depicted in the lower panel. Scale bars=10 μm.
	Figure 3. Importin β inhibits the NabKin–NTE interaction with F-actin, RanGTP counteracts the effect. (A) PtK2 cells expressing the NTE of NabKin (XlNabKinaa1-268), HsKIF14 (HsKIF14aa1-345) or XtKIF14 (XtKIF14aa1-341) fused to the N terminus of EGFP were fixed, stained for F-actin and DNA, and analysed by CLSM. Line plots represent pixel values along the dashed line. Note the correlation between NTE (green) and F-actin signals (red). The colocalisation might even be underestimated, because the assay neglects the avidity contribution from a potential coiled-coil-mediated dimerisation of the kinesins. Scale bar=10 μm. (B) Immobilised NabKin–NTE was incubated with diluted Xenopus oocyte extract in the absence or presence of 2 μM RanQ69L·GTP (a GTPase-deficient mutant; Bischoff et al, 1994). Following a washing step, bound proteins were eluted with buffer containing 5 μM RanQ69L·GTP to increase specificity of elution, and were analysed by SDS–PAGE and Coomassie staining (see also Supplementary Figure S4B). (C) Import of NabKin–NTE fused to EGFP into the nuclei of digitonin-permeabilised HeLa cells was performed for 10 min at RT. Reactions were stopped by fixation and examined by CLSM. Addition of Importin β facilitates nuclear import of NabKin–NTE–EGFP. Scale bar=10 μm. (D) NabKin–NTE–EGFP (2 μM) was assayed for actin-bundling activity (see also Figure 2C). Where indicated, 2 μM Importin-β was also present. The image was cut at black lines and rearranged for clarity. (E) Equal amounts of phalloidin beads were incubated in an undiluted high-speed egg extract and allowed to bind F-actin complexes in the absence or presence of 20 μM RanQ69L·GTP. Bound fractions were immunoblotted with indicated antibodies. RanGTP stimulated binding of NabKin, but not of Capping protein alpha.
	Figure 4. NabKin interacts with meiotic microtubule structures and cortical F-actin after NEBD. Oocytes were fixed at various stages of maturation, stained as detailed in Materials and Methods, and were analysed by CLSM as whole mounts of hemisections along the animal–vegetal axis to follow NabKin localisation during this process. Cartoons explain the analysed structures (see also Supplementary Figure S7). Scale bars A–D=50 μm. (A) Prophase I-arrested stage VI oocyte. (B) Formation of the disc-shaped transient microtubule array directly following NEBD. (C) Transient microtubule array moving towards cortex. (D) MI spindle formation at cortex. (E) High magnification of another MI spindle. Note that NabKin colocalises with the spindle and specifically also with individual microtubules extending from the spindle (indicated by arrows). Scale bar=5 μm.
	Figure 5. NabKin localisation during PB extrusion. CLSM analysis of first and second PB extrusion. Oocytes and eggs were fixed and stained as detailed in the Materials and Methods section. Matured oocytes (A–C) were analysed as whole mounts of hemisections along the animal–vegetal axis. Arrested (D) or activated (E, F) eggs were analysed as whole mounts. Scale bars=10 μm. (A) MI spindle attached to the oocyte cortex. High-sensitivity settings allowed detection of NabKin at the spindle and the cortex. The signal of NabKin at the spindle is usually weaker than the cortical signal. (B) Cortical F-actin cap located directly above the MI spindle during anaphase. (C) CLSM analysis of a nascent first PB. A maximum intensity projection of sections spanning 11.7 μm in depth is shown. (D) Egg arrested in metaphase of meiosis II. High-sensitivity settings allowed detection of NabKin at the MII spindle and the already extruded first PB. (E) A Ca2+-ionophore-activated egg extruding the second PB, stained for NabKin, F-actin and DNA. A maximum intensity projection of sections spanning 34.6 μm in depth is shown. Note that the NabKin signal is far stronger at the nascent PB II compared to the first PB I. (F) Another activated egg extruding the second PB, stained also for a marker of the actomyosin ring (myosin light chain 2). Note that NabKin colocalises with F-actin at the ‘roof' of the PB as well as with the actomyosin ring structure. A maximum intensity projection of sections spanning 3.8 μm in depth is shown.
	Figure 6. KIF14 localisation during mitotic cell division in live HeLa cells. (A) Confocal time-lapse micrographs of live HeLa cells expressing mRFP-α-tubulin and KIF14-mEGFP. Arrowheads indicate cortical pool of KIF14, arrows indicate central spindle. (B) Confocal time-lapse micrographs of live HeLa cells expressing LifeAct-mCherry, which we used to visualise F-actin (Riedl et al, 2008) and KIF14-mEGFP. Arrowheads indicate cortical pool of KIF14, arrows indicate central spindle. Time indicated (s) is relative to first image. Scale bars=10 μm.
	Figure 7. Interaction of NabKin with F-actin is crucial for PB extrusion. (A) Equal amounts of phalloidin beads were incubated in undiluted high-speed extract from Xenopus eggs and allowed to bind F-actin complexes in the absence or presence of 50 μg/ml anti-NabKin–NTE Fab fragment. 20 μM RanQ69L·GTP was added to the extract to release inhibitory Importin β from the NabKin–NTE (see also Figure 3E). Bound fractions were immunoblotted with indicated antibodies. Addition of anti-NabKin–NTE Fab fragment dramatically reduced the amount of NabKin bound to the matrix, but not the amount of bound Capping protein alpha. (B) CLSM analysis of PB I extrusion. Oocytes received nuclear injections with 50 ng of indicated Fab fragments. Oocytes were fixed 3 h post NEBD to allow sufficient time for completion of meiosis I and arrest in metaphase of meiosis II (Gard et al, 1995; Castro et al, 2003). Only oocytes displaying the spindle at the cortex were included in the analysis, as this location marks the site of the PB extrusion. Note that these anti-NabKin–NTE Fab fragment-injected oocytes do not show an extruded PB. Instead, two spindles or an enlarged spindle with two metaphase chromosome plates (indicated by red arrows in lowest panel) were found in these depicted oocytes. Scale bar=10 μm. See Supplementary Figure S6D for additional analyses. (C) Statistical analysis of successful PB extrusion. Uninjected oocytes (59% extruded PB I; n=32; actual oocyte numbers are shown in the graph bars) and anti-GFP-Fab fragment-injected oocytes (53%; n=30) showed no significant difference (NS; P=0.63; χ2-test). The injected Fab fragment against the NabKin–NTE (8%; n=49) impaired PB extrusion highly significantly (***P<0.0001; χ2-test) as compared to uninjected or control injected cells.

References [+] :

Ankenbauer, Proteins regulating actin assembly in oogenesis and early embryogenesis of Xenopus laevis: gelsolin is the major cytoplasmic actin-binding protein. 1988, Pubmed, Xenbase