Evidence for LINC1-SUN associations at the plant nuclear periphery

Abstract

Sad1/UNC84 (SUN) domain proteins are a highly conserved family of inner nuclear membrane localised proteins in eukaryotes. One of their main functions is as key components of nucleo-cytoskeletal bridging complexes, in which SUN proteins associate with nucleoskeletal elements. In metazoans these are the lamins, which form a supportive structural network termed the lamina. Plants lack sequence homologs of lamins but have a similar nucleoplasmic structural network to support the plant NE. Putative components of this plant lamina-like structure are Little Nuclei (LINC) proteins, which bear structural resemblance to lamins and fulfil similar functions. This work explores the associations between AtLINC1, AtSUN1 and AtSUN2. AtLINC1 is recruited to the NE by SUN proteins and is immobilised therein. This recruitment and the immobile properties are likely due to AtSUN1/2-AtLINC1 protein interactions occurring in planta. In addition, the SUN N-terminus appears to play an important role in mediating these interactions. The associations between AtLINC1 and plant SUN proteins are a first indicator of how the nucleoskeleton may be anchored to the nuclear membrane in plants. Building on the previous characterisation of Klarsicht/Anc1/Syne1 homology (KASH) like proteins in plants, this study advances the identification and characterisation of nucleo-cytoskeletal bridging complexes in plants.

Figure 1. Subcellular localisation of AtLINC1-YFP; transient expression of AtLINC1-YFP results in three types of localisation: A) only nucleoplasmic, B) nucleoplasmic with nuclear periphery accumulation, C) only nuclear periphery; D) AtLINC1-YFP (green) remains localised at the nuclear periphery upon co-expression with AtSUN1-CFP and AtSUN2-CFP (magenta), which are present at the nuclear envelope.

However, NE-localised CFP-AtSUN1 and CFP-AtSUN2 cause AtLINC1-YFP to remain nucleoplasmic indicating that the SUN protein N-terminus is involved in recruitment of AtLINC1-YFP to the nuclear periphery; E) AtSUN2^1–106-CFP and AtLINC1-YFP co-localise in the nucleoplasm.

Figure 2. Changes in AtLINC1-YFP localisation; Percentage of expressing cells, in which AtLINC1-YFP is localised in the nucleoplasm only (blue), in both nucleoplasm and at the nuclear periphery (red), and only at the nuclear periphery (green).

A) AtLINC1-YFP localisation upon co-expression with AtSUN1 constructs; B) AtLINC1-YFP localisation upon co-expression with AtSUN2 constructs; number of nuclei analysed = approx. 75 per sample.

Figure 3. FRAP recovery master curves (relate to table 2); AtLINC1-YFP has a low recovery at the plant NE (black curves; A) AtLINC1-YFP mobility when co-expressed with AtSUN1 constructs; B) AtLINC1-YFP mobility when co-expressed with AtSUN2 constructs; full length AtSUN1-CFP/AtSUN2-CFP reduce the recovery of AtLINC1-YFP (red curves) but when the SUN N-terminus is deleted, AtLINC1-YFP recovery increases (blue curves); C) Representative image of FRAP experiment – LINC1-YFP fluorescence at the NE is bleached in ROI (red circle) and recovery monitored; size bar = 10μm.