Mammalian Septins Nomenclature

There are 10 known mammalian septin genes, some of which produce multiple splice variants. The current nomenclature for the genes and gene products is very confusing, with several different names having been given to the same gene product and distinct names given to splice variants of the same gene. Moreover, some names are based on those of yeast or Drosophila septins that are not the closest homologues. Therefore, we suggest that the mammalian septin field adopt a common nomenclature system, based on that adopted by the Mouse Genomic Nomenclature Committee and accepted by the Human Genome Organization Gene Nomenclature Committee. The human and mouse septin genes will be named SEPT1–SEPT10 and Sept1–Sept10, respectively. Splice variants will be designated by an underscore followed by a lowercase “v” and a number, e.g., SEPT4_v1

A systems analysis of importin-α–β mediated nuclear protein import

Importin-β (Impβ) is a major transport receptor for Ran-dependent import of nuclear cargo. Impβ can bind cargo directly or through an adaptor such as Importin-α (Impα). Factors involved in nuclear transport have been well studied, but systems analysis can offer further insight into regulatory mechanisms. We used computer simulation and real-time assays in intact cells to examine Impα–β-mediated import. The model reflects experimentally determined rates for cargo import and correctly predicts that import is limited principally by Impα and Ran, but is also sensitive to NTF2. The model predicts that CAS is not limiting for the initial rate of cargo import and, surprisingly, that increased concentrations of Impβ and the exchange factor, RCC1, actually inhibit rather than stimulate import. These unexpected predictions were all validated experimentally. The model revealed that inhibition by RCC1 is caused by sequestration of nuclear Ran. Inhibition by Impβ results from depletion nuclear RanGTP, and, in support of this mechanism, expression of mRFP-Ran reversed the inhibition

The adapter importin-α provides flexible control of nuclear import at the expense of efficiency

Although there exists a large family of nuclear transport receptors (Karyopherins), the majority of known import cargoes use an adapter protein, Importin-α (Impα), which links the cargo to a karyopherin, Importin-β (Impβ). The reason for the existence of transport adapters is unknown. One hypothesis is that, as Impα re-export is coupled to GTP hydrolysis, it can drive a higher concentration of nuclear cargo than could be achieved by direct cargo binding to Importin-β. However, computer simulations predicted the opposite outcome, and showed that direct transport is faster than adapter-mediated transport. These predictions were validated experimentally. The data, together with previous analyses of nuclear protein import, suggest that the use of adapters such as importin-α provides the cell with increased dynamic range for control of nuclear import rates, but at the expense of efficiency

Par-3 mediates the inhibition of LIM kinase 2 to regulate cofilin phosphorylation and tight junction assembly

The polarity protein Par-3 plays critical roles in axon specification and the establishment of epithelial apico-basal polarity. Par-3 associates with Par-6 and atypical protein kinase C and is required for the proper assembly of tight junctions, but the molecular basis for its functions is poorly understood. We now report that depletion of Par-3 elevates the phosphorylated pool of cofilin, a key regulator of actin dynamics. Expression of a nonphosphorylatable mutant of cofilin partially rescues tight junction assembly in cells lacking Par-3, as does the depletion of LIM kinase 2 (LIMK2), an upstream kinase for cofilin. Par-3 binds to LIMK2 but not to the related kinase LIMK1. Par-3 inhibits LIMK2 activity in vitro, and overexpressed Par-3 suppresses cofilin phosphorylation that is induced by lysophosphatidic acid. Our findings identify LIMK2 as a novel target of Par-3 and uncover a molecular mechanism by which Par-3 could regulate actin dynamics during cell polarization

Regulation of chromatin binding by a conformational switch in the tail of the Ran exchange factor RCC1

RCC1 is the only known exchange factor for the Ran guanosine triphosphatase and performs essential roles in nuclear transport, spindle organization, and nuclear envelope formation. RCC1 binds to chromatin through a bimodal attachment to DNA and histones, and defects in binding cause chromosome missegregation. Chromatin binding is enhanced by apo-Ran. However, the mechanism underlying this regulation has been unclear. We now demonstrate that the N-terminal tail of RCC1 is essential for association with DNA but inhibits histone binding. Apo-Ran significantly promotes RCC1 binding to both DNA and histones, and these effects are tail mediated. Using a fluorescence resonance energy transfer biosensor, we detect conformational changes in the tail of RCC1 coupled to the two binding modes and in response to interactions with Ran and importin-α. The biosensor also reports changes accompanying mitosis in living cells. We propose that Ran induces an allosteric conformational switch in the tail that exposes the histone-binding surface on RCC1 and facilitates association of the positively charged tail with DNA

Exportin-5, a novel karyopherin, mediates nuclear export of double-stranded RNA binding proteins

We have identified a novel human karyopherin (Kap)β family member that is related to human Crm1 and the Saccharomyces cerevisiae protein, Msn5p/Kap142p. Like other known transport receptors, this Kap binds specifically to RanGTP, interacts with nucleoporins, and shuttles between the nuclear and cytoplasmic compartments. We report that interleukin enhancer binding factor (ILF)3, a double-stranded RNA binding protein, associates with this Kap in a RanGTP-dependent manner and that its double-stranded RNA binding domain (dsRBD) is the limiting sequence required for this interaction. Importantly, the Kap interacts with dsRBDs found in several other proteins and binding is blocked by double-stranded RNA. We find that the dsRBD of ILF3 functions as a novel nuclear export sequence (NES) in intact cells, and its ability to serve as an NES is dependent on the expression of the Kap. In digitonin-permeabilized cells, the Kap but not Crm1 stimulated nuclear export of ILF3. Based on the ability of this Kap to mediate the export of dsRNA binding proteins, we named the protein exportin-5. We propose that exportin-5 is not an RNA export factor but instead participates in the regulated translocation of dsRBD proteins to the cytoplasm where they interact with target mRNAs

Importance of the Rab3a-GTP Binding Domain for the Intracellular Stability and Function of Rabphilin3a in Secretion

We had previously demonstrated that Rab3a-GTP inhibits and the Rab3a-binding protein Rabphilin3a enhances secretion in bovine chromaffin cells. In this study, we investigated the role of Rab3a-GTP binding in the intracellular expression and the function of Rabphilin3a in regulated exocytosis in bovine chromaffin cells. Using transient transfections, we found that a minimal domain, Rp(51 190), that inhibits secretion coincides with a minimal domain that effectively binds Rab3a-GTP and allows intracellular stability of the construct. This domain includes a cysteine-rich, Zn 2+ -binding domain whose integrity is also required for Rab3a-GTP binding and the ability to inhibit secretion. A Rabphilin3a mutant, containing both C2 domains but defective in Rab3a-GTP, and wild-type Rabphilin3a both localized to chromaffin granules and stimulated secretion similarly despite lessened intracellular expression of the mutant protein. The data are consistent with a sequence of events in which a Rab3a-GTP Rabphilin3a complex forms on the secretory granule as a precursor in a pathway that enhances secretion. The complex dissociates (perhaps because of GTP hydrolysis) to permit the enhancement of secretion by Rabphilin3a.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66356/1/j.1471-4159.1997.69010164.x.pd

The PAR Proteins: Fundamental Players in Animal Cell Polarization

The par genes were discovered in genetic screens for regulators of cytoplasmic partitioning in the early embryo of C. elegans, and encode six different proteins required for asymmetric cell division by the worm zygote. Some of the PAR proteins are localized asymmetrically and form physical complexes with one another. Strikingly, the PAR proteins have been found to regulate cell polarization in many different contexts in diverse animals, suggesting they form part of an ancient and fundamental mechanism for cell polarization. Although the picture of how the PAR proteins function remains incomplete, cell biology and biochemistry are beginning to explain how PAR proteins polarize cells

LGN Blocks the Ability of NuMA to Bind and Stabilize Microtubules A Mechanism for Mitotic Spindle Assembly Regulation

AbstractLGN is closely related to a Drosophila protein, Partner of inscuteable (Pins), which is required for polarity establishment and asymmetric cell divisions during embryonic development [1–3]. In mammalian cells, LGN binds with high affinity to the C-terminal tail of NuMA, a large nuclear protein that is required for spindle organization, and accumulates at the spindle poles during mitosis [4–9]. LGN also regulates spindle organization, possibly through inhibition of NuMA function [10], but the mechanism of this effect has not yet been understood. Using mammalian cells, frog egg extracts, and in vitro assays, we now show that a small domain within the C terminus of NuMA stabilizes microtubules (MTs), and that LGN blocks stabilization. The nuclear localization signal adjacent to this domain is not involved in stabilization. NuMA can interact directly with MTs, and the MT binding domain on NuMA overlaps by ten amino acid residues with the LGN binding domain. We therefore propose that a simple steric exclusion model can explain the inhibitory effect of LGN on NuMA-dependent mitotic spindle organization

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin–Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to βPIX. Rather, Scrib depletion disrupts E-cadherin–mediated cell–cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin–α-catenin fusion protein but not by E-cadherin–green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration