Inherited nuclear pore substructures template post-mitotic pore assembly.

Nuclear envelope assembly during late mitosis includes rapid formation of several thousand complete nuclear pore complexes (NPCs). This efficient use of NPC components (nucleoporins or "NUPs") is essential for ensuring immediate nucleocytoplasmic communication in each daughter cell. We show that octameric subassemblies of outer and inner nuclear pore rings remain intact in the mitotic endoplasmic reticulum (ER) after NPC disassembly during prophase. These "inherited" subassemblies then incorporate into NPCs during post-mitotic pore formation. We further show that the stable subassemblies persist through multiple rounds of cell division and the accompanying rounds of NPC mitotic disassembly and post-mitotic assembly. De novo formation of NPCs from newly synthesized NUPs during interphase will then have a distinct initiation mechanism. We postulate that a yet-to-be-identified modification marks and "immortalizes" one or more components of the specific octameric outer and inner ring subcomplexes that then template post-mitotic NPC assembly during subsequent cell cycles.

HDAC6 mediates an aggresome-like mechanism for NLRP3 and pyrin inflammasome activation.

Inflammasomes are supramolecular complexes that play key roles in immune surveillance. This is accomplished by the activation of inflammatory caspases, which leads to the proteolytic maturation of interleukin 1ß (IL-1ß) and pyroptosis. Here, we show that nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3)- and pyrin-mediated inflammasome assembly, caspase activation, and IL-1ß conversion occur at the microtubule-organizing center (MTOC). Furthermore, the dynein adapter histone deacetylase 6 (HDAC6) is indispensable for the microtubule transport and assembly of these inflammasomes both in vitro and in mice. Because HDAC6 can transport ubiquitinated pathological aggregates to the MTOC for aggresome formation and autophagosomal degradation, its role in NLRP3 and pyrin inflammasome activation also provides an inherent mechanism for the down-regulation of these inflammasomes by autophagy. This work suggests an unexpected parallel between the formation of physiological and pathological aggregates.

Dynamics of Auxilin 1 and GAK in clathrin-mediated traffic.

Clathrin-coated vesicles lose their clathrin lattice within seconds of pinching off, through the action of the Hsc70 "uncoating ATPase." The J- and PTEN-like domain-containing proteins, auxilin 1 (Aux1) and auxilin 2 (GAK), recruit Hsc70. The PTEN-like domain has no phosphatase activity, but it can recognize phosphatidylinositol phosphate head groups. Aux1 and GAK appear on coated vesicles in successive transient bursts, immediately after dynamin-mediated membrane scission has released the vesicle from the plasma membrane. These bursts contain a very small number of auxilins, and even four to six molecules are sufficient to mediate uncoating. In contrast, we could not detect auxilins in abortive pits or at any time during coated pit assembly. We previously showed that clathrin-coated vesicles have a dynamic phosphoinositide landscape, and we have proposed that lipid head group recognition might determine the timing of Aux1 and GAK appearance. The differential recruitment of Aux1 and GAK correlates with temporal variations in phosphoinositide composition, consistent with a lipid-switch timing mechanism.

Creating novel translation inhibitors to target pro-survival proteins in chronic lymphocytic leukemia.

The viability of chronic lymphocytic leukemia (CLL) is critically dependent upon staving off death by apoptosis, a hallmark of CLL pathophysiology. The recognition that Mcl-1, a major component of the anti-apoptotic response, is intrinsically short-lived and must be continually resynthesized suggested a novel therapeutic approach. Pateamine A (PatA), a macrolide marine natural product, inhibits cap-dependent translation by binding to the initiation factor eIF4A. In this study, we demonstrated that a synthetic derivative of PatA, des-methyl des-amino PatA (DMDAPatA), blocked mRNA translation, reduced Mcl-1 protein and initiated apoptosis in CLL cells. This action was synergistic with the Bcl-2 antagonist ABT-199. However, avid binding to human plasma proteins limited DMDAPatA potency, precluding further development. To address this, we synthesized a new series of PatA analogs and identified three new leads with potent inhibition of translation. They exhibited less plasma protein binding and increased cytotoxic potency toward CLL cells than DMDAPatA, with greater selectivity towards CLL cells over normal lymphocytes. Computer modeling analysis correlated their structure-activity relationships and suggested that these compounds may act by stabilizing the closed conformation of eIF4A. Thus, these novel PatA analogs hold promise for application to cancers within the appropriate biological context, such as CLL.

Dynamics of phosphoinositide conversion in clathrin-mediated endocytic traffic.

Vesicular carriers transport proteins and lipids from one organelle to another, recognizing specific identifiers for the donor and acceptor membranes. Two important identifiers are phosphoinositides and GTP-bound GTPases, which provide well-defined but mutable labels. Phosphatidylinositol and its phosphorylated derivatives are present on the cytosolic faces of most cellular membranes. Reversible phosphorylation of its headgroup produces seven distinct phosphoinositides. In endocytic traffic, phosphatidylinositol-4,5-biphosphate marks the plasma membrane, and phosphatidylinositol-3-phosphate and phosphatidylinositol-4-phosphate mark distinct endosomal compartments. It is unknown what sequence of changes in lipid content confers on the vesicles their distinct identity at each intermediate step. Here we describe 'coincidence-detecting' sensors that selectively report the phosphoinositide composition of clathrin-associated structures, and the use of these sensors to follow the dynamics of phosphoinositide conversion during endocytosis. The membrane of an assembling coated pit, in equilibrium with the surrounding plasma membrane, contains phosphatidylinositol-4,5-biphosphate and a smaller amount of phosphatidylinositol-4-phosphate. Closure of the vesicle interrupts free exchange with the plasma membrane. A substantial burst of phosphatidylinositol-4-phosphate immediately after budding coincides with a burst of phosphatidylinositol-3-phosphate, distinct from any later encounter with the phosphatidylinositol-3-phosphate pool in early endosomes; phosphatidylinositol-3,4-biphosphate and the GTPase Rab5 then appear and remain as the uncoating vesicles mature into Rab5-positive endocytic intermediates. Our observations show that a cascade of molecular conversions, made possible by the separation of a vesicle from its parent membrane, can label membrane-traffic intermediates and determine their destinations.

Recruitment dynamics of ESCRT-III and Vps4 to endosomes and implications for reverse membrane budding.

The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.

Miro1-mediated mitochondrial positioning shapes intracellular energy gradients required for cell migration.

It has long been postulated, although never directly demonstrated, that mitochondria are strategically positioned in the cytoplasm to meet local requirements for energy production. Here we show that positioning of mitochondria in mouse embryonic fibroblasts (MEFs) determines the shape of intracellular energy gradients in living cells. Specifically, the ratio of ATP to ADP was highest at perinuclear areas of dense mitochondria and gradually decreased as more-peripheral sites were approached. Furthermore, the majority of mitochondria were positioned at the ventral surface of the cell, correlating with high ATP:ADP ratios close to the ventral membrane, which rapidly decreased toward the dorsal surface. We used cells deficient for the mitochondrial Rho-GTPase 1 (Miro1), an essential mediator of microtubule-based mitochondrial motility, to study how changes in mitochondrial positioning affect cytoplasmic energy distribution and cell migration, an energy-expensive process. The mitochondrial network in Miro1-/- MEFs was restricted to the perinuclear area, with few mitochondria present at the cell periphery. This change in mitochondrial distribution dramatically reduced the ratio of ATP to ADP at the cell cortex and disrupted events essential for cell movement, including actin dynamics, lamellipodia protrusion, and membrane ruffling. Cell adhesion status was also affected by changes in mitochondrial positioning; focal adhesion assembly and stability was decreased in Miro1-/- MEFs compared with Miro1+/+  MEFs. Consequently Miro1-/- MEFs migrated slower than control cells during both collective and single-cell migration. These data establish that Miro1-mediated mitochondrial positioning at the leading edge provides localized energy production that promotes cell migration by supporting membrane protrusion and focal adhesion stability.

Membrane dynamics of dividing cells imaged by lattice light-sheet microscopy.

Membrane remodeling is an essential part of transferring components to and from the cell surface and membrane-bound organelles and for changes in cell shape, which are particularly critical during cell division. Earlier analyses, based on classical optical live-cell imaging and mostly restricted by technical necessity to the attached bottom surface, showed persistent formation of endocytic clathrin pits and vesicles during mitosis. Taking advantage of the resolution, speed, and noninvasive illumination of the newly developed lattice light-sheet fluorescence microscope, we reexamined their assembly dynamics over the entire cell surface and found that clathrin pits form at a lower rate during late mitosis. Full-cell imaging measurements of cell surface area and volume throughout the cell cycle of single cells in culture and in zebrafish embryos showed that the total surface increased rapidly during the transition from telophase to cytokinesis, whereas cell volume increased slightly in metaphase and was relatively constant during cytokinesis. These applications demonstrate the advantage of lattice light-sheet microscopy and enable a new standard for imaging membrane dynamics in single cells and multicellular assemblies.

Deeply diverged alleles in the Arabidopsis AREB1 transcription factor drive genome-wide differences in transcriptional response to the environment.

Gene regulatory variation is an important driver of the evolution of physiological and developmental responses to the environment. The abscisic acid (ABA) signaling pathway has long been studied as a key component of the cellular response to abiotic stresses in plants. We identify two haplotypes in an Arabidopsis thaliana transcription factor, AREB1, which plays a central role in ABA-mediated response to osmotic stress. These two haplotypes show the sequence signature of long-term maintenance of genetic diversity, suggesting a role for a diversifying selection process such as balancing selection. We find that the two haplotypes, distinguished by a large number of single nucleotide polymorphisms and the presence or absence of four small insertion/deletions in AREB1 intron 1 and exon 2, are at roughly equal frequencies in Arabidopsis, and show high linkage disequilibrium and deep sequence divergence. We use a transgenic approach, along with mRNA Sequencing-based assay of genome-wide expression levels, and find considerable functional divergence between alleles representing the two haplotype groups. Specifically, we find that, under benign soil-water conditions, transgenic lines containing different AREB1 alleles differ in the expression of a large number of genes associated with pathogen response. There are relatively modest gene expression differences between the two transgenic lines under restricted soil water content. Our finding of pathogen-related activity expands the known roles of AREB1 in A. thaliana and reveals the molecular basis of gene-by-environment interaction in a putatively adaptive plant regulatory protein.