Terence D Allen

HIV‐1 DNA Flap formation promotes uncoating of the pre‐integration complex at the nuclear pore

The HIV‐1 central DNA Flap acts as a cis‐acting determinant of HIV‐1 genome nuclear import. Indeed, DNA‐Flap re‐insertion within lentiviral‐derived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV‐1 nuclear import. Here, we show that reverse transcription (RT°) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate post‐fusion event, but rather occurs at the nuclear pore upon RT° completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV‐1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV‐1 RT°, acts as a viral promoting element for the uncoating of HIV‐1 at the nuclear pore.

The nucleoporin Nup153 is required for nuclear pore basket formation, nuclear pore complex anchoring and import of a subset of nuclear proteins

The nuclear pore complex (NPC) is a large proteinaceous structure through which bidirectional transport of macromolecules across the nuclear envelope (NE) takes place. Nup153 is a peripheral NPC component that has been implicated in protein and RNP transport and in the interaction of NPCs with the nuclear lamina. Here, Nup153 is localized by immunogold electron microscopy to a position on the nuclear ring of the NPC. Nuclear reconstitution is used to investigate the role of Nup153 in nucleo‐ cytoplasmic transport and NPC architecture. NPCs assembled in the absence of Nup153 lacked several nuclear basket components, were unevenly distributed in the NE and, unlike wild‐type NPCs, were mobile within the NE. Importin α/β‐mediated protein import into the nucleus was strongly reduced in the absence of Nup153, while transportin‐mediated import was unaffected. This was due to a reduction in import complex translocation rather than to defective receptor recycling. Our results therefore reveal functions for Nup153 in NPC assembly, in anchoring NPCs within the NE and in mediating specific nuclear import events.

Structural and functional organization of the nuclear envelope

The double-membrane nuclear envelope is punctuated by pores where the two membranes are joined. These pores are stabilized by the elaborate nuclear pore complex, which is anchored to the inner membrane by the nuclear lamina, as well as to other nuclear and cytoskeletal structures. Recent experiments have identified proteins involved in the stability of this organization as well as in the function of the nuclear pore complex, which we relate here to newer aspects of nuclear envelope structure.

Ultrastructure of cell loss in intestinal mucosa.

Small intestinal crypt cell production is balanced by an equal cell loss from the villus. In mouse small intestine this cell loss appears to be largely achieved by the extrusion of individual cells by a process of intercellular vacuolation resulting in detachment of the entire cell. This process is similar to that involved in the extrusion of effete goblet cells, apoptotic cells, and the migration of individual epidermal basal cells (reported elsewhere). Scanning electron microscopy reveals a sequence of surface changes concomitant with the stages of cell loss. Though not clearly defined, there was some suggestion of circadian variations in the level of cell loss.

Desmosomal form, fate, and function in mammalian epidermis

Separation of epidermal desmosomal contact is achieved by two means. Proliferative cells which both migrate and divide change their cell to cell contact by an isolation and cytoplasmic separation followed by engulfment of the entire desmosome. During desquamation of cornified cells contacts are broken by an apparent enzymatic degradation mediated via the membrane coating granules (MCG). Differences in this process can be observed according to the rate of cell production. In slowly proliferating regions (ear and dorsal skin) the “stacked” cornified cells have their desmosomes replaced by a specialised peripheral band of attachment, a squamosome ; in regions with a higher proliferative rate (foot) the “nonstacked” cornified cells retain many of their desmosomes.

The fragile X: a scanning electron microscope study.

Scanning electron microscopy (SEM) has been used to study the fragile X chromosome. The fragile site appears as an isochromatid gap in the majority of cases, confirming light microscope (LM) observations. SEM has allowed a more precise location of the fragile site to the Xq27 . 3 region.

Inn1 couples contraction of the actomyosin ring to membrane ingression during cytokinesis in budding yeast.

By rapidly depleting each of the essential budding yeast proteins of unknown function, we identified a novel factor that we call Inn1, which associates with the contractile actomyosin ring at the end of mitosis and is needed for cytokinesis. We show that Inn1 has a C2 domain at the amino terminus of the protein that is required for ingression of the plasma membrane, whereas the remainder of the protein recruits Inn1 to the actomyosin ring. The lethal effects of deleting the INN1 gene can be suppressed by artificial fusion of the C2 domain to other components of the actomyosin ring, restoring membrane ingression on contraction of the actomyosin ring. Our data indicate that recruitment of the C2 domain of Inn1 to the contractile actomyosin ring is crucial for ingression of the plasma membrane during cytokinesis in budding yeast.

Scanning electron microscopy of the G-banded human karyotype.

Abstract The chromosome structure of human metaphases was observed in the scanning electron microscope (SEM) after exposure to G-banding techniques for light microscopy (LM). Individual chromosomes showed an inherent specificity of quaternary coiling. Circumferential grooves along the chromatids demarcated the individual gyres of the coils, which were shown to correspond to the LM G-banding pattern. An increased number of quaternary coils was observed in prometaphase chromosomes, which were shown to be correlated with the high resolution LM bands. We propose that the observation of G-bands relies on LM visualization of quaternary structure by accumulation of Giemsa stain between the coils.

The contraction of collagen matrices by dermal fibroblasts

Floating collagen gel cultures containing human foreskin fibroblasts have been observed to undergo a rapid contraction process. The initial rate of contraction (i.e., within the first 2 hr) was observed to be a linear function of cell number within the concentration range of 10(5)-10(6) cells/gel. Observation of thick, deresined sections of such contracting gels in the SEM, as well as observation of thin sections in the TEM, suggest that the fibroblasts exert a tension upon the surrounding collagen fibers. These observations further indicate that the fibroblasts migrate from the interior regions of the gel matrix and eventually form a monolayer of cells encapsulating the contracted collagen disc. These observations are discussed in terms of the possible mechanisms involved in gel contraction.

Concentration of Ran on chromatin induces decondensation, nuclear envelope formation and nuclear pore complex assembly.

Nuclear envelope (NE) formation can be studied in a cell-free system made from Xenopus eggs. In this system, NE formation involves the small GTPase Ran. Ran associates with chromatin early in nuclear assembly and concentration of Ran on inert beads is sufficient to induce NE formation. Here, we show that Ran binds to chromatin prior to NE formation and recruits RCC1, the nucleotide exchange factor that generates Ran-GTP. In extracts prepared by high-speed centrifugation, increased concentrations of Ran are sufficient to induce chromatin decondensation and NE assembly. Using field emission in-lens scanning electron microscopy (FEISEM), we show that Ran promotes the formation of smoothed membranes and the assembly of nuclear pore complexes (NPCs). In contrast, RanT24N, a mutant that fails to bind GTP and inhibits RCC1, does not support efficient NE assembly, whereas RanQ69L, a mutant locked in a GTP-bound state, permits some membrane vesicle recruitment to chromatin, but inhibits vesicle fusion and NPC assembly. Thus, binding of Ran to chromatin, followed by local generation of Ran-GTP and GTP hydrolysis by Ran, induces chromatin decondensation, membrane vesicle recruitment, membrane formation and NPC assembly. We propose that the biological activity of Ran is determined by its targeting to structures such as chromatin as well as its guanine nucleotide bound state.