The nuclear envelope (NE) aids in organizing the interphase genome by tethering chromatin to the nuclear periphery. During mitotic entry, NE–chromatin contacts are broken. Here, we report on the consequences of impaired NE removal from chromatin for cell division of human cells. Using a membrane–chromatin tether that cannot be dissociated when cells enter mitosis, we show that a failure in breaking membrane–chromatin interactions impairs mitotic chromatin organization, chromosome segregation and cytokinesis, and induces an aberrant NE morphology in postmitotic cells. In contrast, chromosome segregation and cell division proceed successfully when membrane attachment to chromatin is induced during metaphase, after chromosomes have been singularized and aligned at the metaphase plate. These results indicate that the separation of membranes and chromatin is critical during prometaphase to allow for proper chromosome compaction and segregation. We propose that one cause of these defects is the multivalency of membrane–chromatin interactions.

The nuclear compartment is delimited by a specialized expanded sheet of the endoplasmic reticulum (ER) known as the nuclear envelope (NE). Compared to the outer nuclear membrane and the contiguous peripheral ER, the inner nuclear membrane (INM) houses a unique set of transmembrane proteins that serve a staggering range of functions. Many of these functions reflect the exceptional position of INM proteins at the membrane-chromatin interface. Recent research revealed that numerous INM proteins perform crucial roles in chromatin organization, regulation of gene expression, genome stability, and mediation of signaling pathways into the nucleus. Other INM proteins establish mechanical links between chromatin and the cytoskeleton, help NE remodeling, or contribute to the surveillance of NE integrity and homeostasis. As INM proteins continue to gain prominence, we review these advancements and give an overview on the functional versatility of the INM proteome.

Newly synthesized membrane proteins are targeted to the inner nuclear membrane (INM) by diffusion within the membrane system of the endoplasmic reticulum (ER), translocation through nuclear pore complexes (NPCs) and retention on nuclear partners. Using a visual in vitro assay we previously showed that efficient protein targeting to the INM depends on nucleotide hydrolysis. We now reveal that INM targeting is GTP-dependent. Exploiting in vitro reconstitution and in vivo analysis of INM targeting, we establish that Atlastins, membrane-bound GTPases of the ER, sustain the efficient targeting of proteins to the INM by their continued activity in preserving ER topology. When ER topology is altered, the long-range diffusional exchange of proteins in the ER network and targeting efficiency to the INM are diminished. Highlighting the general importance of proper ER topology, we show that Atlastins also influence NPC biogenesis and timely exit of secretory cargo from the ER.

Eukaryotic cells have 2 to 3 discrete nucleoli required for ribosome synthesis. Nucleoli are phase separated nuclear sub-organelles. Here we examined the role of nuclear Lamins and nucleolar factors in modulating the compartmentalization and dynamics of histone 2B (H2B-ECFP) in the nucleolus. Live imaging and Fluorescence Recovery After Photobleaching (FRAP) of labelled H2B, showed that the depletion of Lamin B1, Fibrillarin (FBL) or Nucleostemin (GNL3), enhances H2B-ECFP mobility in the nucleolus. Furthermore, Nucleolin knockdown significantly decreases H2B-ECFP compartmentalization in the nucleolus, while H2B-ECFP residence and mobility in the nucleolus was prolonged upon Nucleolin overexpression. Co-expression of N-terminal and RNA binding domain (RBD) deletion mutants of Nucleolin or inhibiting 45S rRNA synthesis reduces the sequestration of H2B-ECFP in the nucleolus. Taken together, these studies reveal a crucial role of Nucleolin-rRNA complex in modulating the compartmentalization, stability and dynamics of H2B within the nucleolus.

Newly synthesized membrane proteins are inserted into the endoplasmic reticulum (ER) from where they are constantly sorted to various cellular compartments. To analyze and visualize sorting of membrane proteins to the inner nuclear membrane (INM), we developed a trap-release system that uncouples membrane integration into the ER from transport. This assay allows the simultaneous release of a large pool of an INM-destined membrane protein from the ER and microscopy-based monitoring of targeting to the INM. The use of semi-permeabilized HeLa cells further enables the identification and characterization of essential requirements of the targeting process. This protocol provides a detailed description of reporter construction, in vitro reconstitution, and visualization of trafficking.