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Biomedical subjects

M W Goldberg

Publications and source records attributed to M W Goldberg.

At least 19 recordsLinked to original sources
PubMed2001

Steps of nuclear pore complex disassembly and reassembly during mitosis in early Drosophila embryos.

The mechanisms of nuclear pore complex (NPC) assembly and disassembly during mitosis in vivo are not well defined. To address this and to identify the steps of the NPC disassembly and assembly, we investigated Drosophila embryo nuclear structure at the syncytial stage of early development using field emission scanning electron microscopy (FESEM), a high resolution surface imaging technique, and transmission electron microscopy. Nuclear division in syncytial embryos is characterized by semi-closed mitosis, during which the nuclear membranes are ruptured only at the polar regions and are arranged into an inner double membrane surrounded by an additional 'spindle envelope'. FESEM analysis of the steps of this process as viewed on the surface of the dividing nucleus confirm our previous in vitro model for the assembly of the NPCs via a series of structural intermediates, showing for the first time a temporal progression from one intermediate to the next. Nascent NPCs initially appear to form at the site of fusion between the mitotic nuclear envelope and the overlying spindle membrane. A model for NPC disassembly is offered that starts with the release of the central transporter and the removal of the cytoplasmic ring subunits before the star ring.

Animals↗
PubMed2000

Ran alters nuclear pore complex conformation.

Transport across the nuclear membranes occurs through the nuclear pore complex (NPC), and is mediated by soluble transport factors including Ran, a small GTPase that is generally GDP-bound during import and GTP-bound for export. The dynamic nature of the NPC structure suggests a possible active role for it in driving translocation. Here we show that RanGTP but not RanGDP causes alterations of NPC structure when injected into the cytoplasm of Xenopus oocytes, including compaction of the NPC and extension of the cytoplasmic filaments. RanGTP caused accumulation of nucleoplasmin-gold along the length of extended cytoplasmic filaments, whereas RanGDP caused accumulation around the cytoplasmic rim of the NPC. This suggests a possible role for Ran in altering the conformation of the cytoplasmic filaments during transport.

Amino Acid Substitution↗
PubMed2000

The nuclear pore complex: mediator of translocation between nucleus and cytoplasm.

The enclosure of nuclear contents in eukaryotes means that cells require sites in the boundary that mediate exchange of material between nucleus and cytoplasm. These sites, termed nuclear pore complexes (NPCs), number 100-200 in yeast, a few thousand in mammalian cells and approximately 50 million in the giant nuclei of amphibian oocytes. NPCs are large (125 MDa) macromolecular complexes that comprise 50-100 different proteins in vertebrates. In spite of their size and complex structure, NPCs undergo complete breakdown and reformation at cell division. Transport through NPCs can be rapid (estimated at several hundred molecules/pore/second) and accommodates both passive diffusion of relatively small molecules, and active transport of complexes up to several megadaltons in molecular mass. Each pore can facilitate both import and export. The two processes apparently involve multiple pathways for different cargoes, and their transport signals, transport receptors and adapters, and the molecules (and their regulators) that underpin the transport mechanisms. Over the past few years there has been an increasing interest in the pore complex: structural studies have been followed by elucidation of the biochemical aspects of nuclear import, and subsequent investigations into nuclear export. The current challenge is to understand the interactions between the structural elements of the pore complex and the mechanisms that drive the physical processes of translocation through it.

Animals↗
PubMed2000

The nuclear pore complex: structure, function, and dynamics.

A full understanding of nucleocytoplasmic transport depends on knowledge of nuclear pore complex (NPC) structure, the functional roles of NPC components, their interactions during transport and dynamics during the cell cycle. NPC structure is conserved, flexible, and is not simply a tunnel between the nucleus and cytoplasm but appears to be actively involved in the transport process by a series of structural modifications. Transport through the NPC begins in either of its asymmetrical peripheral compartments that are both structurally reorganized during transport in different ways. The central compartment is composed of two symmetrical halves, and functions as a system of transiently open, discrete gates that is not believed to play a role in determining direction. Each NPC subunit has a specific morphology that corresponds to the functional role it plays. A complicated system of vertical and horizontal connections may allow one part of the NPC to transmit a signal to other parts, leading to an ordered series of conformational changes that drive translocation. High-resolution scanning electron microscopy has identified sequential stages of NPC assembly in vitro and revealed how the individual NPC components are assembled into a mature NPC. This review focuses on structural events during transport and on possible mechanisms of NPC assembly.

Animals↗
PubMed1998

Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy.

At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.

Animals↗
PubMed1998

Active nuclear pore complexes in Chironomus: visualization of transporter configurations related to mRNP export.

The Nuclear Pore Complex (NPC) regulates nucleocytoplasmic transport by providing small channels for passive diffusion and multiple docking surfaces that lead to a central translocation channel for active transport. In this study we have investigated by high resolution scanning and transmission electron microscopy the dynamics of NPC structure in salivary gland nuclei from Chironomus during Balbiani ring (BR) mRNP translocation, and present evidence of rearrangement of the transporter related to mRNP export. Analysis of the individual NPC components verified a strong evolutionary conservation of NPC structure between vertebrates and invertebrates. The transporter is an integral part of the NPC and is composed of a central short double cylinder that is retained within the inner spoke ring, and two peripheral globular assemblies which are tethered to the cytoplasmic and nucleoplasmic coaxial rings by eight conserved internal ring filaments. Distinct stages of BR mRNP nuclear export through the individual NPC components were directly visualized and placed in a linear transport sequence. The BR mRNP first binds to the NPC basket, which forms an expanded distal basket ring. In this communication we present stages of BR mRNP transport through the nucleoplasmic, central and cytoplasmic transporter subunits, which change their conformation during mRNP translocation, and the emergence of mRNP into the cytoplasm. We propose that the reorganization of the basket may be driven, in part, by an active translocation process at the transporter. Furthermore, the images provide dramatic evidence that the transporter functions as a central translocation channel with transiently open discrete gates in its globular assemblies. A model of NPC transporter reorganization accompanied with mRNP translocation is discussed.

Animals↗
PubMed1997

Three-dimensional visualization of the route of protein import: the role of nuclear pore complex substructures.

The three-dimensional localization of nucleoplasmin and wheat germ agglutinin (WGA) at the nuclear envelope of Xenopus oocytes is demonstrated by microinjecting protein coated gold colloids and examining their distribution using both stereo transmission electron microscopy and field emission in-lens scanning electron microscopy. Localization of many WGA gold particles and nucleoplasmin gold particles at the same nuclear pore complex (NPC) following coinjection is demonstrated. Binding of the WGA gold in the central region of the NPCs appears to form a barrier, preventing the import of nucleoplasmin gold, and includes central localization along radial "tracks" which correspond to the internal filaments connecting the cytoplasmic ring and the central region of the NPC. We suggest that these filaments may in some way be involved in opening and closing of the central channel of the NPC for transport. Transport of nucleoplasmin through the central region of the NPCs appears to be in "single file" regardless of the size of the colloidal gold, and distribution into the nucleoplasm appears to be through the basket rings with no association of the nucleoplasmin gold with the basket filaments being observed.

Animals↗
PubMed1997

Macromolecular substructure in nuclear pore complexes by in-lens field-emission scanning electron microscopy.

Scanning electron microscopy (SEM) has produced a wealth of novel images that have significantly complemented our perception of biological structure and function, derived initially from transmission electron microscopy (TEM) information. SEM is a surface imaging technology, and its impact at the subcellular level has been restricted by reduced resolution in comparison with TEM. Recently, SEM resolution has been considerably improved by the advent of high-brightness sources used in field-emission instruments (FEISEM) which have produced resolution of around 1 nm, virtually equivalent to TEM "working resolution." Here we review our findings in the use of FEISEM in the imaging of nuclear envelopes and their associated structures, such as nuclear pore complexes, and the relationships of structure and function. FEISEM allows the structurally orientated cell biologist to visualise, directly and in three dimensions, subcellular structure and its modulation with a view to understanding its functional significance.

Animals↗
PubMed1997

Nuclear pore complex structure in birds.

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.

Animals↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗
PubMed1997

Xenopus Ran-binding protein 1: molecular interactions and effects on nuclear assembly in Xenopus egg extracts.

Ran is a nuclear GTPase implicated in nucleocytoplasmic transport, the maintenance of nuclear structure, mRNA processing, and cell cycle regulation. By two-hybrid interaction in yeast, we have identified a Xenopus homologue of Ran-binding protein 1 (RanBP1). Xenopus RanBP1 interacts specifically with the GTP-bound form of Ran and forms complexes in Xenopus egg extracts with Ran, importin-beta/karyopherin-beta and importin-alpha/karyopherin-alpha, but not p10, p120/RanBP7, RanBP2 or other nucleoporins. These complexes may play roles in the recycling of Ran and importins/karyopherins during nucleocytoplasmic transport. Increased concentrations of RanBP1 stabilise an interaction between Ran and RCC1 in egg extracts, inhibiting the exchange activity of RCC1 towards Ran. Under these conditions, the assembly of nuclei from chromatin is dramatically affected: the nuclei do not assemble a lamina and become very small with homogeneously condensed chromatin. They fail to actively import proteins and do not undergo DNA replication. By field emission in-lens scanning electron microscopy, we show that these nuclei have an intact nuclear envelope containing pore complexes, but the envelope is highly convoluted. However, RanBP1 does not directly inhibit nuclear protein import in assembled nuclei. These results suggest that RCC1 and/or Ran have a function early in nuclear assembly that is disrupted by RanBP1.

Amino Acid Sequence↗
PubMed1997

Dimples, pores, star-rings, and thin rings on growing nuclear envelopes: evidence for structural intermediates in nuclear pore complex assembly.

We used field emission in-lens scanning electron microscopy to examine newly-assembled, growing nuclear envelopes in Xenopus egg extracts. Scattered among nuclear pore complexes were rare 'dimples' (outer membrane depressions, 5-35 nm diameter), more abundant holes (pores) with a variety of edge geometries (35-45 nm diameter; 3.3% of structures), pores containing one to eight triangular 'star-ring' subunits (2.1% of total), and more complicated structures. Neither mature complexes, nor these novel structures, formed when wheat germ agglutinin (which binds O-glycosylated nucleoporins) was added at high concentrations (>500 microg/ml) directly to the assembly reaction; low concentrations (10 microg/ml) had no effect. However at intermediate concentrations (50-100 microg/ml), wheat germ agglutinin caused a dramatic, sugar-reversible accumulation of 'empty' pores, and other structures; this effect correlated with the lectin-induced precipitation of a variable proportion of each major Xenopus wheat-germ-agglutinin-binding nucleoporin. Another inhibitor, dibromo-BAPTA (5,5'-dibromo-1,2-bis[o-aminophenoxylethane-N,N,N',N'-tetraacetic acid), had different effects depending on its time of addition to the assembly reaction. When 1 mM dibromo-BAPTA was added at time zero, no pore-related structures formed. However, when dibromo-BAPTA was added to growing nuclei 40-45 minutes after initiating assembly, star-rings and other structures accumulated, suggesting that dibromo-BAPTA can inhibit multiple stages in pore complex assembly. We propose that assembly begins with the formation and stabilization of a hole (pore) through the nuclear envelope, and that dimples, pores, star-rings, and thin rings are structural intermediates in nuclear pore complex assembly.

Animals↗
PubMed1996

RNP export is mediated by structural reorganization of the nuclear pore basket.

Messenger RNA leaves the cell nucleus as ribonucleoprotein (RNP) particles. The nucleocytoplasmic translocation of the particles takes place through the nuclear pore complex (NPC) and includes two steps: binding to the NPC and transit through its central channel. The NPC basket is a fishtrap-like component of NPC facing the nucleoplasm. Its position in the NPC strongly suggests that it has an important role in the initial steps of macromolecular export from the nucleus. Here we report a cyclic rearrangement of the basket structure in relation to the translocation of a specific messenger RNP (mRNP) of exceptional size, the Balbiani ring RNP particles in the salivary gland cells in Chironomus. We used field emission in-lens scanning electron microscopy (FEISEM), transmission electron microscopy (TEM), and immunocytochemistry to analyse the structural organization of the basket during the mRNP export. Our observations reveal five configurations of the basket which are presented in a model of basket reorganization related to the state of mRNP penetration into the NPC. We suggest that the functional role of the basket is to anchor the mRNP particle to the NPC and position it in correct orientation at the entrance to the central channel of the NPC.

Animals↗
PubMed1996

The nuclear pore complex and lamina: three-dimensional structures and interactions determined by field emission in-lens scanning electron microscopy.

Three dimensional surface imaging was used to examine structural details of the nuclear pore complex. Subsurface structures were uncovered by detergent extraction, proteolysis, mechanical fracturing and combinations of these. Features observed in this way were mostly consistent with previous three-dimensional structures with some novel observations. In addition to cytoplasmic and basket filaments attached to each coaxial ring, we have observed radiating filaments within the central channel. New details of basket organization are presented, showing that basket filaments branch and are woven together to form the basket ring. The "central transporter" is shown to be a regular, consistent structure revealed after removal of overlying internal filaments. The lumenal ring is visualised and we present evidence that the lamina may be attached to the spoke ring complex. Finally we show that there is evidence for a novel structure, the "star ring", sandwiched between the cytoplasmic ring and the membrane.

Animals↗
PubMed1996

Nuclear lamina and nuclear matrix organization in sperm pronuclei assembled in Xenopus egg extract.

Nuclear lamina and matrices were prepared from sperm pronuclei assembled in Xenopus egg extracts using a fractionation and extraction procedure. Indirect immunofluorescence revealed that while chromatin was efficiently removed from nuclei during the extraction procedure, the distribution of lamins was unaffected. Consistent with this data, the amount of lamin B3, determined by immunoblotting, was not affected through the extraction procedure. Nuclear matrices were visualised in DGD sections by TEM. Within these sections filaments were observed both at the boundary of the nucleus (the lamina) and within the body of the nucleus (internal nuclear matrix filaments). To improve resolution, nuclear matrices were also prepared as whole mounts and viewed using field emission in lens scanning electron microscopy (FEISEM). This technique revealed two distinct networks of filaments. Filaments lying at the surface of nuclear matrices interconnected nuclear pores. These filaments were readily labelled with monoclonal anti-lamin B3 antibodies. Filaments lying within the body of the nuclear matrix were highly branched but were not readily labelled with antilamin B3 antibodies. Nuclear matrices were also prepared from sperm pronuclei assembled in lamin B3 depleted extracts. Using FEISEM, filaments were also detected in these preparations. However, these filaments were poorly organised and often appeared to aggregate. To confirm these results nuclear matrices were also observed as whole mounts using TEM. Nuclear matrices prepared from control nuclei contained a dense array of interconnected filaments. Many (but not all) of these filaments were labelled with anti-lamin B3 antibodies. In contrast, nuclear matrices prepared from "lamin depleted nuclei' contained poorly organised or aggregated filaments which were not specifically labelled with anti-lamin B3 antibodies.

Animals↗