The Supramolecular Organization of the C. elegans Nuclear Lamin Filament

Abstract

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.

Introduction

The nuclear lamina is a meshwork layer apposed to the inner nuclear membrane of all metazoan cell nuclei.¹ This layer provides structural support and constitutes a functional interface between the nuclear envelope and the peripheral chromatin.2, 3 The major molecular constituents of the nuclear lamina are the nuclear lamins.³ On the basis of their amino acid sequence, the nuclear lamins have been classified as type V intermediate filament (IF) proteins and are believed to be the ancestors of cytoplasmic IF proteins.4, 5 Physical interactions between lamins and other cytoskeletal elements, including actin filaments, microtubules and cytoplasmic IFs, are mediated by inner and outer nuclear membrane protein complexes.⁶

Like all IF proteins, lamins consist of a central α-helical coiled-coil forming a rod domain, flanked by a non-α-helix amino-terminal head domain and a partially globular carboxy-terminal tail domain (Fig. 1a).⁷ The rod domain consists of four coiled-coil-forming α-helical segments, termed 1A, 1B, 2A and 2B, each of which is made of periodic heptad repeats. The lamin tail contains a nuclear localization signal and a highly conserved 105 amino acid region that contains an immunoglobulin (Ig)-fold. The head domain is a short domain that is not predicted to fold into a distinct conformation.⁸

Electron microscopy has provided snapshots of the in vitro assembly of lamins, thereby revealing the initial steps involved in the formation of higher-order lamin supramolecular structures. Accordingly, the elementary building block of all lamins is a polar, ∼ 52 nm long rod-like dimer, flanked at one end by two globular tail domains packed in close proximity.⁹ Thus, the two central α-helical rod domains within a lamin dimer are coiled around each other in a parallel, unstaggered fashion. In a first assembly step, lamin dimers elongate “head-to-tail” with a short overlap to yield polar polymers exhibiting a 48 nm axial repeat (Fig. 1c).¹⁰ These polar head-to-tail polymers of lamin dimers can further associate laterally and eventually form lamin filaments (Fig. 2f) and paracrystalline fibres (Fig. 3e and f).9, 11

As yet, the physical conditions necessary for in vitro assembly into stable IF-like 10 nm filaments have not been determined for most lamins. On the other hand, a characteristic feature of all lamins is their propensity to form paracrystalline fibres exhibiting a distinct axial repeat pattern in vitro.11, 12 While the physiological significance of these higher-order assemblies remains elusive, they have been shown to form in insect cells upon over-expression of Xenopus or Drosophila lamins.¹³ Despite extensive morphological characterization of the different lamin assemblies, the 3D supramolecular organization of lamin dimers within lamin filaments and paracrystalline fibres has not been determined to date.

On the basis of their primary amino acid sequence, lamins are classified as A- or B-type. The B-type lamins are expressed in all cells, remain associated with the membranes during mitosis, and are known to be the major component of the nuclear lamina before breakdown of the nuclear envelope.¹⁴ Caenorhabditis elegans contains a single B-type lamin gene, termed lmn-1, encoding the Ce-lamin protein.15, 16 Compared to vertebrate lamins, the coil 2B segment found within the central α-helical rod domain of the Ce-lamin is shortened by two heptad repeats, and lacks the SPTR sequence upstream of coil 1A, presenting a phosphorylation site recognized by the mitotic kinase Cdk1.¹⁷ Moreover, Ce-lamin has one of the shortest tail domains (179 amino acids in length) of any known lamin. Remarkably, as yet, Ce-lamin is the only lamin that can be assembled in vitro into stable IF-like filaments, a hallmark of all known cytoplasmic IF proteins, including the invertebrate isoforms.9, 18

A prerequisite for deciphering the 3D supramolecular organization of lamins in situ is to dissect their in vitro assembly. Towards this goal, we have determined the 3D organization of the Ce-lamin protein within the in vitro-assembled IF-like filament and paracrystalline fibres, in the present work. First, we analyzed filamentous lamin by cryo-electron tomography (cryo-ET) and determined the 3D structural organization of lamin dimers depicted within distinct IF-like filaments. More specifically, we document that IF-like lamin filaments are composed of three or four tetrameric protofilaments, each containing two anti-parallel, partially staggered head-to-tail polymers of dimers. Second, we examined the 3D structure of paracrystalline lamin fibres by applying averaging procedures to cryo-electron tomograms containing these distinct higher-order lamin assemblies. Most significantly, the 3D organization of anti-parallel head-to-tail polymers of lamin dimers within the paracrystalline fibres is very similar to that found in the IF-like filaments. Third, we show that a disease-causing mutation alters the structure of IF-like lamin filaments. Taken together, these results reveal, for the first time, the 3D molecular organization of distinct in vitro lamin assemblies thereby opening an avenue for systematic investigation of the effects of laminopathic mutations on lamin assembly and supramolecular organization.