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. 2015;6(2):144-53.
doi: 10.1080/19491034.2014.1003512. Epub 2015 Mar 11.

Plant nuclear shape is independently determined by the SUN-WIP-WIT2-myosin XI-i complex and CRWN1

Xiao Zhou  1 , Norman Reid Groves, Iris Meier
Affiliations

Plant nuclear shape is independently determined by the SUN-WIP-WIT2-myosin XI-i complex and CRWN1

Xiao Zhou et al. Nucleus. 2015.

Abstract

Nuclei undergo dynamic shape changes during plant development, but the mechanism is unclear. In Arabidopsis, Sad1/UNC-84 (SUN) proteins, WPP domain-interacting proteins (WIPs), WPP domain-interacting tail-anchored proteins (WITs), myosin XI-i, and CROWDED NUCLEI 1 (CRWN1) have been shown to be essential for nuclear elongation in various epidermal cell types. It has been proposed that WITs serve as adaptors linking myosin XI-i to the SUN-WIP complex at the nuclear envelope (NE). Recently, an interaction between Arabidopsis SUN1 and SUN2 proteins and CRWN1, a plant analog of lamins, has been reported. Therefore, the CRWN1-SUN-WIP-WIT-myosin XI-i interaction may form a linker of the nucleoskeleton to the cytoskeleton complex. In this study, we investigate this proposed mechanism in detail for nuclei of Arabidopsis root hairs and trichomes. We show that WIT2, but not WIT1, plays an essential role in nuclear shape determination by recruiting myosin XI-i to the SUN-WIP NE bridges. Compared with SUN2, SUN1 plays a predominant role in nuclear shape. The NE localization of SUN1, SUN2, WIP1, and a truncated WIT2 does not depend on CRWN1. While crwn1 mutant nuclei are smooth, the nuclei of sun or wit mutants are invaginated, similar to the reported myosin XI-i mutant phenotype. Together, this indicates that the roles of the respective WIT and SUN paralogs have diverged in trichomes and root hairs, and that the SUN-WIP-WIT2-myosin XI-i complex and CRWN1 independently determine elongated nuclear shape. This supports a model of nuclei being shaped both by cytoplasmic forces transferred to the NE and by nucleoplasmic filaments formed under the NE.

Keywords: Arabidopsis; CDS, coding sequence; CRWN; CRWN1, CROWDED NUCLEI 1; KASH; KASH, Klarsicht/ANC-1/Syne-1 Homology; LINC; LINC, linker of the nucleoskeleton to the cytoskeleton; NE, nuclear envelope; NLI, nuclear envelope localization index; SUN; SUN, Sad1/UNC-84; WIP, WPP domain-interacting protein; WIT, WPP domain-interacting tail-anchored protein; XI-iC642, myosin XI-i C-terminal 642 amino acids.; nuclear envelope; nuclear shape; sun1-KO sun2-KD, sun1-knockout sun2-knockdown.

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Figures

Figure 1.
Figure 1.
WIT2 is essential for the elongated nuclear shape in root hairs and trichomes. (A) Root hair and trichome nuclear morphology of wild type, wit1-1 wit2-1, wit1-1, wit2-1, and WIT2pro::GFP-WIT2*-transformed wit2-1. Nuclei of WIT2pro::GFP-WIT2*-transformed wit2-1 were viewed using confocal microscopy. For all others, fluorescence microscopy was used to image Hoechst 33342-stained nuclei, and the trichome nuclei were imaged directly using bright field microscopy. Scale bar equals 10 μm. Images are at the same magnification scale. (B) Root hair nuclear shape quantified by the length of the nuclei. Single asterisk, 0.05 > P > 0.01 when compared to wild type. Double asterisks, P < 0.01 when compared to wild type. Double dots, P < 0.01 when compared to wit1-1 wit2-1. Two-tailed t-test was used and n = 80. Error bars represent SE. (C) Nuclear circularity index of trichome nuclei. The ratio of width and length of the maximum nuclear cross section was used as the index. Double asterisks, P < 0.01, and “O,” P > 0.05, when compared to wild type. Two-tailed t-test was used and n = 50.Double dots, P < 0.01 when compared to wit1-1 wit2-1. Error bars represent SE.
Figure 2.
Figure 2.
SUN1 plays a predominant role in nuclear shape determination. (A) Root hair and trichome nuclear morphology of sun-1-1, sun1-KO, sun2-1, and sun1-KO sun2-KD. Fluorescence microscopy was used to image Hoechst 33342-stained nuclei, and the trichome nuclei were imaged using bright field microscopy. Scale bar equals 10 μm. Images are at the same magnification scale. (B) Root hair nuclear shape quantified by the length of the nuclei. Double asterisks, P < 0.01, when compared to wild type. Double dots, P < 0.01 when compared to sun1-KO sun2-KD. The P value of the t-test between sun2-1 and Ws-4 is indicated in the figure. Two-tailed t-test was used and n = 80. Error bars represent SE. (C) Nuclear circularity index of trichome nuclei. The ratio of width and length of the maximum nucleus cross section was used as the index. Double asterisks, P < 0.01, when compared to wild type. “O,” P > 0.05 when compared to sun1-KO sun2-KD. The P value of the t-test between sun2-1 and Ws-4 is indicated in the figure. Two-tailed t-test was used and n = 50. Error bars represent SE.
Figure 3.
Figure 3.
WIT2 connects myosin XI-i to the SUN-WIP NE bridges.(A) WIT2* interacts with WIP1, WIP2, and WIP3. GFP-tagged proteins were immunoprecipitated and detected with anti-GFP antibody. Myc-tagged proteins were detected with an anti-Myc antibody. Input/IP ratio was 1:9. Numbers on the left indicate molecular mass in kilodaltons. Red arrow heads indicate possible truncated GFP-fusion proteins. (B) WIT1 and WIT2* interacts with XI-iC642. GFP-tagged proteins were immunoprecipitated and detected with anti-GFP antibody. Myc-tagged proteins were detected with an anti-Myc antibody. Input/IP ratio was 1:18. Numbers on the left indicate molecular mass in kilodaltons. Red arrow heads indicate possible truncated GFP-fusion proteins. Vertical back lines represent intervening lanes removed for display purposes. (C) Localization of GFP-XI-iC642 in wild type, sun1-KO sun2-KD, wit1-1, and wit2-1. Scale bar equals 10 μm. All images are single optical sections at the same magnification. (D) NLI of GFP-XI-iC642 in wild type, sun1-KO sun2-KD, wit1-1, and wit2-1. The sum of the 2 maximum NE intensities divided by the sum of the 2 maximum cytoplasmic intensities was used as an NLI (illustrated in the top panel). Double asterisks, P < 0.01, and “O,” P > 0.05, when compared to wild type. Two-tailed t-test was used. For each genotype, 3 transgenic lines and 10 nuclei from each line were analyzed (total n = 30). Error bars represent SE.
Figure 4.
Figure 4.
CRWN1 is not required for the NE-association of the SUN-WIP-WIT2 complex. The localization of SUN1-GFP, SUN2-RFP, GFP-WIP1, and GFP-WIT2* in wild type (left column) and crwn1-1 (middle column) are shown. Scale bar equals 10 μm, and all images are at the same magnification. The NLI are shown in the right column. Asterisk, p = 0.035, and “O,” P > 0.05, when compared to wild type. For each genotype, 3 transgenic lines and 30 nuclei from each line were measured (total n = 90). Error bars represent SE.
Figure 5.
Figure 5.
Root hair nuclear morphology of crwn1-1 is different from sun1-KO sun2-KD and wit1-1 wit2-1. (A) NE morphology illustrated by GFP-WIT1.The nuclei of crwn1-1 are smooth, while the nuclei of sun1-KO sun2-KD and wit1-1 wit2-1 are predominantly invaginated. Scale bar equals 10 μm. All images are at the same magnification. (B) Quantification of smooth nuclei and invaginated nuclei in crwn1-1, sun1-KO sun2-KD, and wit1-1 wit2-1. Double asterisks, P < 0.01, when compared with crwn1-1, 2-tailed Fisher′s exact test. The n of each category is indicated in the figure.
Figure 6.
Figure 6.
Model of nuclear shape determination. Myosin XI-i is anchored to the SUN-WIP NE bridges by WIT2. This SUN-WIP-WIT2-myosin XI-i complex transfers cytoplasmic forces to the NE. In the nucleoplasm, CRWN1 acts independently on nuclear shape determination by forming lamina-like structures. Other nucleoplasmic proteins that affect nuclear shape—CRWN4, KAKU4, and NUP136—and the CRWN1-SUN interaction of unknown function are not drawn.
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