Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2006 Jan 2;172(1):41–53. doi: 10.1083/jcb.200509124

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/).

PMC Copyright notice

Figure 8. — The nesp2G KASH domain interacts with the Sun1 lumenal domain. (A) Fluorescence microscopy of HeLa cells expressing a tetracycline-inducible GFP-KASH fusion protein (HeLaTR GFP-KASH). In the absence of tetracycline (−Tet), GFP-KASH is expressed at low levels and is localized exclusively to the NE. After induction with tetracycline for 24 h (+Tet), GFP-KASH is found throughout the peripheral ER as well as the NE. Expression levels of GFP-KASH within the cell population is extremely uniform both before and after induction. (B) Transfection of SS–HA-Sun1–KDEL into the HeLaTR GFP-KASH cells after tetracycline induction resulted in the complete loss of GFP-KASH from the NE. This was accompanied by the formation of cytoplasmic aggregates (arrows, top). Conversely, introduction of full-length HA-Sun1 into these cells resulted in the recruitment of GFP-KASH to the NE (arrows, middle and bottom). (C) HA-Sun2 was found to have a similar effect (arrows). In the merged images in B and C, GFP-KASH is presented in green, whereas HA-Sun is shown in red. (D) To identify an in vitro interaction between KASH and SUN domains, GFP-KASH, SS–HA-Sun1–KDEL, or both proteins were translated in reticulocyte lysate containing [³⁵S]methionine/cysteine in either the presence or absence of microsomes (Totals). Anti-GFP immunoprecipitation of a fraction of each sample revealed the pull-down of SS–HA-Sun1–KDEL by GFP-KASH when both proteins were cotranslated in the presence of microsomes (arrow). A slightly faster migrating band (asterisk) was detected in the absence of microsomes. However, this band originates in the GFP-KASH translation and is unrelated to HA-Sun1L–KDEL. Molecular masses are indicated in kD. (E) Immunoprecipitation of HeLa cell lysates with anti-Sun2 antibodies coprecipitates a very large anti-nesp2G immunoreactive protein (arrow). HC indicates the position of immunoglobulin heavy chains. These data suggest a significant interaction between KASH and Sun proteins that must involve their lumenal domains. These findings allow us to propose a model for the LINC complex (F) in which nuclear components, including lamins, bind to the INM SUN domain proteins. They, in turn, bind to the KASH domain of the actin-associated giant nesprins on the ONM. Thus, the LINC complex establishes a physical connection between the nucleoskeleton and the cytoskeleton.