Larisa E. Kapinos

Summary: Nuclear transport receptors are best known for mediating nucleocytoplasmic transport (NCT) through nuclear pore complexes. Here, we uncover an unexpected function of the primary import receptor importin-β (Impβ) as a direct regulator of the actin cytoskeleton. Impβ associates with stress fibers and the cell cortex and promotes actin polymerization through direct interactions. Disrupting Impβ-actin binding impairs stress fiber formation and suppresses cell migration well before NCT is affected. In 3D spheroids, perturbing Impβ further compromises tissue integrity, as reflected in changes to nuclear curvature and ellipticity. Together, these findings identify Impβ as a regulator of actin organization in both 2D and 3D contexts, revealing a direct link between the nuclear transport machinery and the cytoskeleton.

Nuclear pore complexes (NPCs) mediate selective exchange of macromolecules between the nucleus and cytoplasm, but the organization of their transport barrier has been a matter of debate. Here we used high-speed atomic force microscopy, complemented with orthogonal in vitro and in vivo approaches, to probe the dynamic behaviour of the NPC central channel at millisecond resolution. We found that nuclear transport factors dynamically remodel intrinsically disordered phenylalanine-glycine (FG) domains tethered within the NPC channel, partitioning the barrier into two zones: a rapidly fluctuating annular region and a highly mobile central plug. Increased FG-repeat density in mutant NPCs dampened barrier dynamics and impaired transport. Notably, NPC-like behaviour was recapitulated in DNA origami nanopores bearing transport factors and correctly tethered FG domains but not in in vitro FG hydrogels. Thus, the rotationally symmetric architecture of NPCs supports a nanoscopic barrier organization that contrasts with many of the bulk properties of in vitro FG-domain assemblies.

Protocells offer a versatile material for dissecting cellular processes and developing simplified biomimetic systems by combining biological components with synthetic ones. However, a gap exists between the integrity and complex functionality of native organelles such as nuclei, and bottom‐up strategies reducing cellular functions within a synthetic environment. Here, this gap is bridged by incorporating native nuclei into polymeric giant unilamellar vesicles (pGUVs) using double‐emulsion microfluidics. It is shown that the nuclei retain their morphology and nuclear envelope integrity, facilitating the import of co‐encapsulated peptide‐based multicompartment micelles (MCMs) via nuclear localization signals (NLS). Importantly, it is demonstrated that the nuclear import machinery remains functional inside the protocells, and by enriching the GUV interior with nuclear import‐promoting factors, the delivery efficiency of NLS‐MCMs significantly increases. The findings reveal that nucleated protocells preserve nuclear function and integrity for extended periods, providing a new platform for studying nuclear processes in a simplified, yet biologically relevant, environment. This approach opens avenues for creating advanced biohybrid materials, offering opportunities to investigate organelle behavior and their interactions with cellular components in greater detail. The findings establish a foundation for high‐throughput applications in synthetic biology and contribute valuable insights into sustaining complex cellular functions in engineered systems.

Nucleoporins (nups) in the nuclear pore complex (NPC) form a selective barrier that suppresses the diffusion of most macromolecules while enabling rapid transport of nuclear transport receptor (NTR)–bound cargos. Recent studies have shown that the NPC may dilate and constrict, but how altering the NPC diameter affects its selective barrier properties remains unclear. Here, we build DNA nanopores with programmable diameters and nup arrangements to model the constricted and dilated NPCs. We find that Nup62 proteins form a dynamic cross-channel barrier impermeable to hepatitis B virus (HBV) capsids when grafted inside 60-nm-wide nanopores but not in 79-nm pores, where Nup62 cluster locally. Furthermore, importin-β1 substantially changes the dynamics of Nup62 assemblies and facilitates the passage of HBV capsids through the 60-nm NPC mimics containing Nup62 and Nup153. Our study shows that transport channel width is critical to the permeability of nup barriers and underscores NTRs’ role in dynamically remodeling nup assemblies and mediating the nuclear entry of viruses.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinβ1 (Kapβ1 or importinβ1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapβ1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

Tau is a neuronal protein that stabilizes axonal microtubules (MTs) in the central nervous system. In Alzheimer’s disease (AD) and other tauopathies, phosphorylated Tau accumulates in intracellular aggregates, a pathological hallmark of these diseases. However, the chronological order of pathological changes in Tau prior to its cytosolic aggregation remains unresolved. These include its phosphorylation and detachment from MTs, mislocalization into the somatodendritic compartment, and oligomerization in the cytosol. Recently, we showed that Tau can interact with phenylalanine-glycine (FG)-rich nucleoporins (Nups), including Nup98, that form a diffusion barrier inside nuclear pore complexes (NPCs), leading to defects in nucleocytoplasmic transport. Here, we used surface plasmon resonance (SPR) and bio-layer interferometry (BLI) to investigate the molecular details of Tau:Nup98 interactions and determined how Tau phosphorylation and oligomerization impact the interactions. Importantly, phosphorylation, but not acetylation, strongly facilitates the accumulation of Tau with Nup98. Oligomerization, however, seems to inhibit Tau:Nup98 interactions, suggesting that Tau-FG Nup interactions occur prior to oligomerization. Overall, these results provide fundamental insights into the molecular mechanisms of Tau-FG Nup interactions within NPCs, which might explain how stress-and disease-associated posttranslational modifications (PTMs) may lead to Tau-induced nucleocytoplasmic transport (NCT) failure. Intervention strategies that could rescue Tau-induced NCT failure in AD and tauopathies will be further discussed.

Nuclear pore complexes (NPCs) discriminate nonspecific macromolecules from importin and exportin receptors, collectively termed “karyopherins” (Kaps), that mediate nucleocytoplasmic transport. This selective barrier function is attributed to the behavior of intrinsically disordered phenylalanine-glycine nucleoporins (FG Nups) that guard the NPC channel. However, NPCs in vivo are typically enriched with different Kaps, and how they impact the NPC barrier remains unknown. Here, we show that two major Kaps, importinβ1/karyopherinβ1 (Kapβ1) and exportin 1/chromosomal maintenance 1 (CRM1), are required to fortify NPC barrier function in vivo. Their enrichment at the NPC is sustained by promiscuous binding interactions with the FG Nups, which enable CRM1 to compensate for the loss of Kapβ1 as a means to maintain NPC barrier function. However, such a compensatory mechanism is constrained by the cellular abundances and different binding kinetics for each respective Kap, as evidenced for importin-5. Consequently, we find that NPC malfunction and nucleocytoplasmic leakage result from poor Kap enrichment.

Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the β-karyopherin (Kapβ) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP–RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapβs, cargoes and Ran to establish the asymmetry of NCT.