Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

In eukaryotes, the double membranous nuclear envelope (NE) encloses the nucleoplasm and separates it from the cytoplasm. The inner nuclear membrane (INM) provides contact with chromatin and the outer nuclear membrane (ONM) is continuous with the endoplasmic reticulum (ER).

The two bilayers are fused at nuclear pore complexes (NPCs) that form aqueous channels through which regulated transport of macromolecules occurs. NPCs consist of multiple copies of ∼30 different nucleoporins (Nups) that are organized into biochemically distinct sub-complexes. Two such modules, the inner ring complex (also called Nup93 complex) and the Y-complex (also called Nup107 complex) constitute the NPC scaffold that is symmetric across the NE plane. FG-Nups (containing phenylalanine-glycine rich intrinsically disordered protein domains) dock onto the scaffold. They constitute the permeability barrier and interact with translocating cargo complexes. Some of them (e.g., Nup214/88, Nup358 [RanBP2], and Nup153) introduce asymmetry by specifically binding to the cytoplasmic or nuclear face of the NPC, respectively.

Obviously, the sheer size and compositional complexity of NPCs renders its assembly and membrane insertion a very intricate task. Two distinct NPC assembly pathways that are temporally separated during the cell cycle have been described. First, during interphase, NPCs are assembled de novo onto an enclosed NE. Interphase assembly occurs ubiquitously throughout eukaryotes and strictly requires the fusion of the INM and ONM by a mechanism that is only partially understood. Second, no membrane fusion is required for NPC assembly at mitotic exit. This so-called postmitotic assembly mode is restricted to eukaryotes that disassemble their NPCs during mitosis into soluble sub-complexes after phosphorylation by mitotic kinases. In anaphase, de-phosphorylation of Nups is thought to trigger the ordered re-assembly onto the separated chromatids before or while membranes enclose daughter nuclei. Both insertion mechanisms rely on the stepwise recruitment of pre-assembled sub-complexes. An insertion of pre-assembled NPCs into the NE has (to the best of our knowledge) not yet been described.

NPCs not only reside within the NE but are also found in stacked cytoplasmic membranes termed annulate lamellae (AL) that are a subdomain of the ER. Based on two-dimensional (2D) transmission electron micrographs these membrane stacks have been perceived as parallel membrane sheets decorated with NPCs (hereafter called AL-NPCs) that morphologically appear similar to their counterparts on the nuclear envelope (NE-NPC). AL appear in some but not all transformed cell lines and are highly abundant in germ cells and early embryos throughout animal phyla, including Xenopus,Caenorhabditis elegans, sea urchin, Drosophila, and also humans.

A role of AL as a storage compartment for maternally deposited Nups that can be made available for meiosis and the rapid cell cycles during early embryogenesis has been suggested but not experimentally proven. Despite these fundamental and long-standing pretensions the function of AL remains elusive and controversial, primarily for two reasons: (1) it has been difficult to conceive how the insertion of parallel stacked membrane sheets containing pre-assembled and possibly pre-oriented NPCs is topologically possible; and (2) direct experimental evidence for a contribution of AL-NPCs to the pool of NE-NPCs has never been obtained. On the contrary a previous study in Drosophila embryos has detected large soluble pools of transport channel Nups and concluded that NPC insertion likely proceeds from soluble cytosolic Nups.

Authors address the function of AL in the physiological context of the Drosophila blastoderm embryo that is rich in AL, while it undergoes a series of 13 synchronized mitoses in a syncytium. Subsequently, the plasma membranes enclose the cortically aligned somatic nuclei in the extended 14thinterphase, forming the first epithelial cell layer before the embryo initiates gastrulation. This occurs concomitantly with the broad onset of transcriptional activity on the zygotic genome, a major developmental transition present in all metazoan.

In the syncytial blastoderm, cell-cycle progression is very rapid, with interphase durations of ∼10 min during the early cell cycles. At least in mammalian cells, de novo NPC interphase assembly has been described to proceed with markedly slower kinetics. This led the authors to hypothesize that NPC assembly into a closed NE in Drosophila embryos might occur by a different, faster mechanism. By tracking NPCs in living embryos, authors demonstrate direct uptake of AL-NPCs into the NE, as the ER feeds nuclear expansion.

They derive a topological model that explains how the INM becomes continuous with inserting membrane sheets from the ER. Authors conclude that AL insertion to the NE is a previously unanticipated mode of NPC insertion that relies on pre-assembled, yet immature NPC scaffolds and operates prior to gastrulation.