Lamin dynamics.

Nuclear lamins form a highly insoluble structure, the nuclear lamina, which is associated with the nuclear envelope. Recent results suggest, however, that the lamins are more dynamic than originally thought. They accumulate in nucleoplasmic foci in the G1 stage of the cell cycle and later appear mainly in the peripheral lamina. Some of the lamin foci are closely associated with heterochromatin. Furthermore, the various lamin types assemble into the lamina polymer independently of each other. Both the assembly and disassembly of the lamins, as well as the interaction of the lamins with other nuclear structures such as the nuclear membrane, may be mediated by phosphorylation and dephosphorylation.

Uptake of HIV-1 tat protein mediated by low-density lipoprotein receptor-related protein disrupts the neuronal metabolic balance of the receptor ligands.

Neurological disorders develop in most people infected with human immunodeficiency virus type 1 (HIV-1). However, the underlying mechanisms remain largely unknown. Here we report that binding of HIV-1 transactivator (Tat) protein to low-density lipoprotein receptor-related protein (LRP) promoted efficient uptake of Tat into neurons. LRP-mediated uptake of Tat was followed by translocation to the neuronal nucleus. Furthermore, the binding of Tat to LRP resulted in substantial inhibition of neuronal binding, uptake and degradation of physiological ligands for LRP, including alpha2-macroglobulin, apolipoprotein E4, amyloid precursor protein and amyloid beta-protein. In a model of macaques infected with a chimeric strain of simian-human immunodeficiency virus, increased staining of amyloid precursor protein was associated with Tat expression in the brains of simian-human immunodeficiency virus-infected macaques with encephalitis. These results indicate that HIV-1 Tat may mediate HIV-1-induced neuropathology through a pathway involving disruption of the metabolic balance of LRP ligands and direct activation of neuronal genes.

Alzheimer-associated presenilins 1 and 2: neuronal expression in brain and localization to intracellular membranes in mammalian cells.

Mutations in two recently identified genes appear to cause the majority of early-onset familial Alzheimer's disease (FAD). These two novel genes, presenilin 1 (PS1) and presenilin 2 (PS2) are members of an evolutionarily conserved gene family. The normal biological role(s) of the presenilins and the mechanism(s) by which the FAD-associated mutations exert their effect remain unknown. Employing in situ hybridization, we demonstrate that the expression patterns of PS1 and PS2 in the brain are extremely similar to each other and that messages for both are primarily detectable in neuronal populations. Immunochemical analyses indicate that PS1 and PS2 are similar in size and localized to similar intracellular compartments (endoplasmic reticulum and Golgi complex). FAD-associated mutations in PS1 and PS2 do not significantly modify either their migration patterns on SDS-polyacrylamide gel electrophoresis or their overall subcellular localization, although subtle differences in perinuclear staining were noted for mutant PS1.

Molecular interactions in paracrystals of a fragment corresponding to the alpha-helical coiled-coil rod portion of glial fibrillary acidic protein: evidence for an antiparallel packing of molecules and polymorphism related to intermediate filament structure.

We have expressed in Escherichia coli a fragment of c-DNA that broadly corresponds to the alpha-helical coiled-coil rod section of glial fibrillary acidic protein (GFAP) and have used the resultant protein to prepare paracrystals in which molecular interactions can be investigated. An engineered fragment of mouse GFAP c-DNA was inserted into a modified version of the E. coli expression vector pLcII, from which large quantities of a lambda cII-GFAP rod fusion protein were prepared. A protein fragment corresponding to the GFAP rod was then obtained by proteolysis with thrombin. Paracrystals of this material were produced using divalent cations (Mg, Ca, Ba) in the presence of a chaotrophic agent such as thiocyanate. These paracrystals showed a number of polymorphic patterns that were based on a fundamental pattern that had dyad symmetry and an axial repeat of 57 nm. Analysis of both positive and negative staining patterns showed that this fundamental pattern was consistent with a unit cell containing two 48-nm-long molecules in an antiparallel arrangement with their NH2 termini overlapping by approximately 34 nm. More complicated patterns were produced by stacking the fundamental pattern with staggers of approximately 1/5, 2/5, and 1/2 the axial repeat. The molecular packing the unit cell was consistent with a range of solution studies on intermediate filaments that have indicated that a molecular dimer (i.e., a tetramer containing four chains or two coiled-coil molecules) is an intermediate in filament assembly. Moreover, these paracrystals allow the molecular interactions involved in the tetramer to be investigated in some detail.

Dynamic properties of nuclear lamins: lamin B is associated with sites of DNA replication.

The nuclear lamins form a fibrous structure, the nuclear lamina, at the periphery of the nucleus. Recent results suggest that lamins are also present as foci or spots in the nucleoplasm at various times during interphase of the cell cycle (Goldman, A. E., R. D. Moir, M. Montag-Lowy, M. Stewart, and R. D. Goldman. 1992. J. Cell Biol. 104:725-732; Bridger, J. M., I. R. Kill, M. O'Farrell, and C. J. Hutchison. 1993. J. Cell Sci. 104:297-306). In this report we demonstrate that during mid-late S-phase, nuclear foci detected with lamin B antibodies are coincident with sites of DNA replication as detected by the colocalization of sites of incorporation of bromodeoxyuridine (BrDU) or proliferating cell nuclear antigen (PCNA). The relationship between lamin B and BrDU is not maintained in the following G1 stage of the cell cycle. Furthermore, the nuclear staining patterns seen with antibodies directed against lamins A and C in mid-late S-phase do not coalign with the lamin B/BrDU-containing structures. These results imply that there is a role for lamin B in the organization of replicating chromatin during S phase.

Motile properties of vimentin intermediate filament networks in living cells.

The motile properties of intermediate filament (IF) networks have been studied in living cells expressing vimentin tagged with green fluorescent protein (GFP-vimentin). In interphase and mitotic cells, GFP-vimentin is incorporated into the endogenous IF network, and accurately reports the behavior of IF. Time-lapse observations of interphase arrays of vimentin fibrils demonstrate that they are constantly changing their configurations in the absence of alterations in cell shape. Intersecting points of vimentin fibrils, or foci, frequently move towards or away from each other, indicating that the fibrils can lengthen or shorten. Fluorescence recovery after photobleaching shows that bleach zones across fibrils rapidly recover their fluorescence. During this recovery, bleached zones frequently move, indicating translocation of fibrils. Intriguingly, neighboring fibrils within a cell can exhibit different rates and directions of movement, and they often appear to extend or elongate into the peripheral regions of the cytoplasm. In these same regions, short filamentous structures are also seen actively translocating. All of these motile properties require energy, and the majority appear to be mediated by interactions of IF with microtubules and microfilaments.

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells.

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.

Pathway of incorporation of microinjected lamin A into the nuclear envelope.

When microinjected into the cytoplasm of 3T3 cells, biotinylated human lamin A rapidly enters the nucleus and gradually becomes incorporated into the nuclear lamina region as determined by immunofluorescence. The incorporation of the microinjected material takes several hours and progresses through a series of morphologically identifiable stages. Within minutes after microinjection, lamin A is found in spots distributed throughout the nucleus, except in nucleolar regions. Over a time course of up to 6 h, these spots appear to decrease in size and number as the biotinylated lamin A becomes associated with the endogenous nuclear lamina. Eventually, the typical nuclear rim staining pattern normally revealed by immunofluorescence with nuclear lamin antibodies is seen with antibiotin. This latter rim staining property is passed on to daughter cells following mitosis. These results indicate that the microinjected biotinylated nuclear lamin A retains those properties required for its integration into the lamina, as well as those necessary for the disassembly and subsequent reassembly of the nuclear lamina during cell division. The initial rapid accumulation into foci and the subsequent slower incorporation into the nuclear lamina appear to be analogous to the stages of incorporation following the microinjection of cytoskeletal intermediate filament proteins such as vimentin and keratin (Vikstrom, K., G. G. Borisy, and R. D. Goldman. 1989. Proc. Natl. Acad. Sci. USA. 86:549-553; Miller, R. K., K. Vikstrom, and R. D. Goldman. 1991. J. Cell Biol. 113:843-855). Foci are also observed in some uninjected cells using nuclear lamin antibodies, indicating that these features are a genuine component of nuclear substructure. Evidence is presented that shows the appearance of these nuclear structures is cell cycle dependent.

Disruption of nuclear lamin organization alters the distribution of replication factors and inhibits DNA synthesis.

The nuclear lamina is a fibrous structure that lies at the interface between the nuclear envelope and the nucleoplasm. The major proteins comprising the lamina, the nuclear lamins, are also found in foci in the nucleoplasm, distinct from the peripheral lamina. The nuclear lamins have been associated with a number of processes in the nucleus, including DNA replication. To further characterize the specific role of lamins in DNA replication, we have used a truncated human lamin as a dominant negative mutant to perturb lamin organization. This protein disrupts the lamin organization of nuclei when microinjected into mammalian cells and also disrupts the lamin organization of in vitro assembled nuclei when added to Xenopus laevis interphase egg extracts. In both cases, the lamina appears to be completely absent, and instead the endogenous lamins and the mutant lamin protein are found in nucleoplasmic aggregates. Coincident with the disruption of lamin organization, there is a dramatic reduction in DNA replication. As a consequence of this disruption, the distributions of PCNA and the large subunit of the RFC complex, proteins required for the elongation phase of DNA replication, are altered such that they are found within the intranucleoplasmic lamin aggregates. In contrast, the distribution of XMCM3, XORC2, and DNA polymerase alpha, proteins required for the initiation stage of DNA replication, remains unaltered. The data presented demonstrate that the nuclear lamins may be required for the elongation phase of DNA replication.

Rapid movements of vimentin on microtubule tracks: kinesin-dependent assembly of intermediate filament networks.

The assembly and maintenance of an extended intermediate filament (IF) network in fibroblasts requires microtubule (MT) integrity. Using a green fluorescent protein-vimentin construct, and spreading BHK-21 cells as a model system to study IF-MT interactions, we have discovered a novel mechanism involved in the assembly of the vimentin IF cytoskeleton. This entails the rapid, discontinuous, and MT-dependent movement of IF precursors towards the peripheral regions of the cytoplasm where they appear to assemble into short fibrils. These precursors, or vimentin dots, move at speeds averaging 0.55 +/- 0.24 micrometer/s. The vimentin dots colocalize with MT and their motility is inhibited after treatment with nocodazole. Our studies further implicate a conventional kinesin in the movement of the vimentin dots. The dots colocalize with conventional kinesin as shown by indirect immunofluorescence, and IF preparations from spreading cells are enriched in kinesin. Furthermore, microinjection of kinesin antibodies into spreading cells prevents the assembly of an extended IF network. These studies provide insights into the interactions between the IF and MT systems. They also suggest a role for conventional kinesin in the distribution of non-membranous protein cargo, and the local regulation of IF assembly.

Disruption of nuclear lamin organization blocks the elongation phase of DNA replication.

The role of nuclear lamins in DNA replication is unclear. To address this, nuclei were assembled in Xenopus extracts containing AraC, a reversible inhibitor that blocks near the onset of the elongation phase of replication. Dominant-negative lamin mutants lacking their NH(2)-terminal domains were added to assembled nuclei to disrupt lamin organization. This prevented the resumption of DNA replication after the release of the AraC block. This inhibition of replication was not due to gross disruption of nuclear envelope structure and function. The organization of initiation factors was not altered by lamin disruption, and nuclei resumed replication when transferred to extracts treated with CIP, an inhibitor of the cyclin-dependent kinase (cdk) 2-dependent step of initiation. This suggests that alteration of lamin organization does not affect the initiation phase of DNA replication. Instead, we find that disruption of lamin organization inhibited chain elongation in a dose-dependent fashion. Furthermore, the established organization of two elongation factors, proliferating cell nuclear antigen, and replication factor complex, was disrupted by DeltaNLA. These findings demonstrate that lamin organization must be maintained in nuclei for the elongation phase of DNA replication to proceed.

Insights into the dynamic properties of keratin intermediate filaments in living epithelial cells.

The properties of keratin intermediate filaments (IFs) have been studied after transfection with green fluorescent protein (GFP)-tagged K18 and/or K8 (type I/II IF proteins). GFP-K8 and -K18 become incorporated into tonofibrils, which are comprised of bundles of keratin IFs. These tonofibrils exhibit a remarkably wide range of motile and dynamic activities. Fluorescence recovery after photobleaching (FRAP) analyses show that they recover their fluorescence slowly with a recovery t(1/2) of approximately 100 min. The movements of bleach zones during recovery show that closely spaced tonofibrils (<1 microm apart) often move at different rates and in different directions. Individual tonofibrils frequently change their shapes, and in some cases these changes appear as propagated waveforms along their long axes. In addition, short fibrils, termed keratin squiggles, are seen at the cell periphery where they move mainly towards the cell center. The motile properties of keratin IFs are also compared with those of type III IFs (vimentin) in PtK2 cells. Intriguingly, the dynamic properties of keratin tonofibrils and squiggles are dramatically different from those of vimentin fibrils and squiggles within the same cytoplasmic regions. This suggests that there are different factors regulating the dynamic properties of different types of IFs within the same cytoplasmic regions.

A role for nuclear lamins in nuclear envelope assembly.

The molecular interactions responsible for nuclear envelope assembly after mitosis are not well understood. In this study, we demonstrate that a peptide consisting of the COOH-terminal domain of Xenopus lamin B3 (LB3T) prevents nuclear envelope assembly in Xenopus interphase extracts. Specifically, LB3T inhibits chromatin decondensation and blocks the formation of both the nuclear lamina-pore complex and nuclear membranes. Under these conditions, some vesicles bind to the peripheral regions of the chromatin. These "nonfusogenic" vesicles lack lamin B3 (LB3) and do not bind LB3T; however, "fusogenic" vesicles containing LB3 can bind LB3T, which blocks their association with chromatin and, subsequently, nuclear membrane assembly. LB3T also binds to chromatin in the absence of interphase extract, but only in the presence of purified LB3. Additionally, we show that LB3T inhibits normal lamin polymerization in vitro. These findings suggest that lamin polymerization is required for both chromatin decondensation and the binding of nuclear membrane precursors during the early stages of normal nuclear envelope assembly.

Dynamic disruptions in nuclear envelope architecture and integrity induced by HIV-1 Vpr.

Human immunodeficiency virus-1 (HIV-1) Vpr expression halts the proliferation of human cells at or near the G2 cell-cycle checkpoint. The transition from G2 to mitosis is normally controlled by changes in the state of phosphorylation and subcellular compartmentalization of key cell-cycle regulatory proteins. In studies of the intracellular trafficking of these regulators, we unexpectedly found that wild-type Vpr, but not Vpr mutants impaired for G2 arrest, induced transient, localized herniations in the nuclear envelope (NE). These herniations were associated with defects in the nuclear lamina. Intermittently, these herniations ruptured, resulting in the mixing of nuclear and cytoplasmic components. These Vpr-induced NE changes probably contribute to the observed cell-cycle arrest.

Clearance of Alzheimer's Abeta peptide: the many roads to perdition.

The amyloid hypothesis of Alzheimer's disease (AD) maintains that the accumulation of the amyloid beta protein (Abeta) is a critical event in disease pathogenesis. A great deal of both academic and commercial research has focused on the mechanisms by which Abeta is generated. However, investigations into the mechanisms underlying Abeta clearance have blossomed over the last several years. This minireview will summarize pathways involved in the removal of cerebral Abeta, including enzymatic degradation and receptor-mediated efflux out of the brain.

The amyloid protein precursor of Alzheimer's disease is a mediator of the effects of nerve growth factor on neurite outgrowth.

The beta A4 protein, the major component of the amyloid deposition characterizing Alzheimer's disease, derives from the amyloid protein precursor (APP), an integral membrane protein with soluble derivatives. The function of APP is unknown. Both soluble and membrane-associated human brain APP (10(-10) M) significantly increased (P less than 0.025) neurite length and branching in pheochromocytoma PC12 cells, but did not affect the number of neurites per cell. At higher concentrations, APP was cytotoxic, with a half-maximal concentration of 5 x 10(-9) M. Nerve growth factor (NGF) is known to affect APP expression in vivo and in vitro. Antibodies to APP specifically diminished the effects of NGF on neurite length and branching. Thus APP may act to mediate neurite outgrowth promotion by NGF.

Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice.

Inhibition of neocortical beta-amyloid (Abeta) accumulation may be essential in an effective therapeutic intervention for Alzheimer's disease (AD). Cu and Zn are enriched in Abeta deposits in AD, which are solubilized by Cu/Zn-selective chelators in vitro. Here we report a 49% decrease in brain Abeta deposition (-375 microg/g wet weight, p = 0.0001) in a blinded study of APP2576 transgenic mice treated orally for 9 weeks with clioquinol, an antibiotic and bioavailable Cu/Zn chelator. This was accompanied by a modest increase in soluble Abeta (1.45% of total cerebral Abeta); APP, synaptophysin, and GFAP levels were unaffected. General health and body weight parameters were significantly more stable in the treated animals. These results support targeting the interactions of Cu and Zn with Abeta as a novel therapy for the prevention and treatment of AD.

Repression of ribosome and tRNA synthesis in secretion-defective cells is signaled by a novel branch of the cell integrity pathway.

The transcription of ribosomal DNA, ribosomal protein (RP) genes, and 5S and tRNA genes by RNA polymerases (Pols) I, II, and III, respectively, is rapidly and coordinately repressed upon interruption of the secretory pathway in Saccharomyces cerevisiae. We find that repression of ribosome and tRNA synthesis in secretion-defective cells involves activation of the cell integrity pathway. Transcriptional repression requires the upstream components of this pathway, including the Wsc family of putative plasma membrane sensors and protein kinase C (PKC), but not the downstream Bck1-Mkk1/2-Slt2 mitogen-activated protein kinase cascade. These findings reveal a novel PKC effector pathway that controls more than 85% of nuclear transcription. It is proposed that the coordination of ribosome and tRNA synthesis with cell growth may be achieved, in part, by monitoring the turgor pressure of the cell.

A tetratricopeptide repeat mutation in yeast transcription factor IIIC131 (TFIIIC131) facilitates recruitment of TFIIB-related factor TFIIIB70.

Transcription factor IIIC (TFIIIC) plays an important role in assembling the initiation factor TFIIIB on genes transcribed by RNA polymerase III (Pol III). In Saccharomyces cerevisiae, assembly of the TFIIIB complex by promoter-bound TFIIIC is thought to be initiated by its tetratricopeptide repeat (TPR)-containing subunit, TFIIIC131, which interacts directly with the TFIIB-related factor, TFIIIB70/Brf1. In this work, we have identified 10 dominant mutations in TFIIIC131 that increase Pol III gene transcription. All of these mutations are found within a discrete 53-amino-acid region of the protein encompassing TPR2. Biochemical studies of one of the mutations (PCF1-2) show that the increase in transcription is due to an increase in the recruitment of TFIIIB70 to TFIIC-DNA. The PCF1-2 mutation does not affect the affinity of TFIIIC for DNA, and the differential in both transcription and TFIIIB complex assembly is observed at saturating levels of TFIIIB70. This indicates that mutant and wild-type TFIIIC-DNA complexes have the same affinity for TFIIIB70 and suggests that the increased recruitment of this factor is achieved by a nonequilibrium binding mechanism. A novel mechanism of activation in which the TPR mutations facilitate a conformational change in TFIIIC that is required for TFIIIB70 binding is proposed. The implications of this model for the regulation of processes involving TPR proteins are discussed.

Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2'-deoxyuridine in the HCT-8 human carcinoma line.

In vitro resistance of HCT-8 cells to 5-fluoro-2'-deoxyuridine (FdUrd) has been obtained after a stepwise increase (up to 1 microM) in the concentration of the nucleoside in the culture medium over a period of 6 months. With a clonogenic assay, the toxicities of 17 antineoplastic agents on HCT-8-sensitive and -resistant cells were compared. Resistant cells were 700-fold resistant to FdUrd and showed different degrees of cross-resistance to several purine and pyrimidine nucleoside analogues; no cross-resistance was noted to base analogues and other cytotoxic drugs. The activities of FdUrd phosphorylase, 5'-fluorouridine kinase, 5-fluorouridine phosphorylase, 5-fluorouracil phosphoribosyltransferase, and thymidylate synthase were not significantly different in the sensitive and resistant cell lines. Mixing experiments indirectly excluded the possible elevation of the level of cytoplasmic phosphatases. The activity of FdUrd kinase in sensitive cell extracts was no more than twice that of resistant cells, and the affinities of this enzyme for FdUrd and thymidine at 0.1 to 50 microM were similar in both cell lines. However, cultures of this line failed to accumulate 5-fluoro-2'-deoxyuridylate at concentrations of FdUrd that resulted in substantial accumulation of the nucleotide in the sensitive line. These contrasting data suggested a defect in the facilitated diffusion of the analogue. The entrance of free nucleoside and its subsequent phosphorylation were compared in the two lines over short (2 to 40 s) and longer time periods at 25 degrees C and at 4 degrees C over a range of extracellular FdUrd concentrations (0.1 to 10 microM). Rapid entrance of the nucleoside into sensitive cells was observed, but entry was not detectable in resistant cells. Dipyridamole and nitrobenzylthioinosine inhibition as well as high-performance liquid chromatography analysis confirmed that data obtained from the sensitive cell line during the first 40 s primarily reflected facilitated diffusion of free nucleoside.