Motifs in Schizosaccharomyces pombe ars3002 important for replication origin activity in Saccharomyces cerevisiae

Ars3002 is an efficient single-copy replication origin in the fission yeast, Schizosaccharomyces pombe. In a previous study, we tested the effects of consecutive approximately 50-bp deletions throughout ars3002 on the replication efficiency of those origins in S. pombe. Here we report the results of our use of the same approximately 50-bp deletions to test the hypothesis that some of the cis-acting sequences important for replication origin activity in fission yeast might be conserved in the evolutionarily distant budding yeast, Saccharomyces cerevisiae. We found that in most cases there was no correlation between the effects of particular mutations in S. pombe and in S. cerevisiae. We conclude that it is unlikely that any of the cis-acting sequences recognised by homologous replication proteins is conserved between these two yeast species.     

Quantification of bacterial adhesion forces using atomic force microscopy (AFM)

This study demonstrated that atomic force microscopy (AFM) can be used to obtain high-resolution topographical images of bacteria, and to quantify the tip-cell interaction force and the surface elasticity. Results show that the adhesion force between the Si3N4 tip and the bacteria surface was in the range from -3.9 to -4.3 nN. On the other hand, the adhesion forces at the periphery of the cell-substratum contact surface ranged from -5.1 to -5.9 nN and those at the cell-cell interface ranged from -6.5 to -6.8 nN. The two latter forces were considerably greater than the former one, most likely due to the accumulation of extracellular polymer substance (EPS). Results also show that the elasticity varied on the cell surface.     

原子力显微镜 (AFM) 在细菌生物被膜研究中的应用

原子力显微镜(AFM)作为一项重要的表面可视化技术,以其独特的优势(纳米级的空间分辨率、皮牛级力灵敏度、免标记、可在溶液环境下工作)被广泛应用于生物被膜的研究。AFM不仅可以在近生理环境下对生物被膜表面超微形貌进行可视化表征,同时还可以通过纳米压痕对生物被膜的机械特性(弹性和粘性)进行定量测量,利用AFM单细胞和单分子力谱技术可以获得生物被膜形成过程中细胞-基底以及细胞-细胞之间的相互作用力,为生物被膜的实时原位系统研究提供了可行性。本文简述了AFM的基本操作原理,综述了近年来AFM用于生物被膜表面超微结构成像、机械特性测量以及相互作用力研究方面的进展,并对AFM在生物被膜研究中面临的问题和未来的发展方向进行了讨论。     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting 'curing' of the adhesive; some force curves also contained a 'fingerprint' of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative 'tanning' mechanism in barnacle cyprid cement.     

An in situ study of the nanomechanical properties of barnacle (Balanus amphitrite) cyprid cement using atomic force microscopy (AFM)

Abstract

Cyprids are the final planktonic stage in the larval dispersal of barnacles and are responsible for surface exploration and attachment to appropriate substrata. The nanomechanical properties of barnacle (Balanus amphitrite) cyprid permanent cement were studied in situ using atomic force microscopy (AFM). Force curves were recorded from the cement disc continually over the course of its curing and these were subsequently analysed using custom software. Results showed a narrowing of the pull-off force distribution with time, as well as a reduction in molecular stretch length over time. In addition, there was a strong correlation between maximum pull-off force and molecular stretch length for the cement, suggesting ‘curing’ of the adhesive; some force curves also contained a ‘fingerprint’ of modular protein unfolding. This study provides the first direct experimental evidence in support of a putative ‘tanning’ mechanism in barnacle cyprid cement.     

Visualization of chromatin folding patterns in chicken erythrocytes by atomic force microscopy （AFM）

The organization of the higher order structure of chromatin in chicken erythrocytes has been examined with tapping-mode scanning force microscopy under conditions close to their native envirinment.Reproducible highresolution AFM images of chromatin compaction at several levels can be demonstrated.An extended beads-on-astring (width of - 15-20nm,height of - 2-3nm for each individual nucleosome) can be consistently observed.Furthermore,superbeade (width of - 40nm,height of - 7nm) are demonstrated.Visualization of the solenoid conformation at the level of 30nm chromatin fiber is attained either by using AFM or by using electron microscopy.In addition,tightly coiled chromatin fibers (- 50-60nm and - 90-110nm) can be revealed.Our data suggest that the chromatin in the interphase nucleus of chicken erythrocyte represents a high-order conformation and AFM provides useful high-resolution structural information concerning the folding pattern of interphase chromatin fibers.     

Annealing and melting behavior of poly(l-lactic acid) single crystals as revealed by in situ atomic force microscopy

In situ annealing and melting of folded-chain single crystals of poly(l-lactic acid) (PLLA) was examined by temperature-controlled atomic force microscopy (AFM). Prominent changes in the crystal appearance during annealing could be followed in real time by the AFM at temperatures above the original crystallization temperature. Thickening of the crystal edges could be occasionally observed, and this indicates that the crystal edges are less perfect than the central, well-ordered regions. At higher annealing temperatures, melting of the unthickened part started. The melting of the unthickened region progressed from the boundaries of the thickened portion normal to the growth face, rather than to the folding surfaces. In addition, it is suggested that melting also initiates at defective or distorted sites in the crystal as revealed by transmission electron microscopy (TEM) and AFM.     

Force and compliance measurements on living cells using atomic force microscopy (AFM)

We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study. Published: January 15, 2004     

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe.     

Analysis of the intrinsic bend in the M13 origin of replication by atomic force microscopy

Atomic force microscopy (AFM) has been used to image a 471-bp bent DNA restriction fragment derived from the M13 origin of replication in plasmid LITMUS 28, and a 476-bp normal, unbent fragment from plasmid pUC19. The most probable angle of curvature of the 471-bp DNA fragment is 40-50 degrees, in reasonably good agreement with the bend angle determined by transient electric birefringence, 38 degrees +/- 7 degrees. The normal 476-bp DNA fragment exhibited a Gaussian distribution of bend angles centered at 0 degrees, indicating that this fragment does not contain an intrinsic bend. The persistence length, P, was estimated to be 60 +/- 8 and 62 +/- 8 nm for the 471- and 476-bp fragments, respectively, from the observed mean-square end-to-end distances in the AFM images. Since the P-values of the normal and bent fragments are close to each other, the overall flexibility of DNA fragments of this size is only marginally affected by the presence of a stable bend. The close agreement of AFM and transient electric birefringence results validates the suitability of both methods for characterizing DNA bending and flexibility.     

Visual representation by atomic force microscopy (AFM) of tomato spotted wilt virus ribonucleoproteins.

Atomic force microscopy (AFM) allows the observation of biological material without fixation procedures. Here we present AFM images of ribonucleoproteins (nucleocapsids) derived from a plant infecting RNA virus (tomato spotted wilt virus, TSWV), which have been recorded in contact mode. The nucleocapsids, prepared from systemically infected leaves of tobacco, were spreaded on a glass surface and dried in air, and appeared as regularly formed rings, resembling the proposed pseudocircular and panhandle structure of encapsidated genomic RNA. Average values between 1300 and 2200 nm of nucleocapsid lengths could be related to dimensions estimated by electron microscopy, thereby validating a filamentous configuration of the TSWV ribonucleoproteins. However, to our knowledge regular, ring-like forms of ribonucleoproteins have not been obtained by electron microscopy, which rather showed an amorphous structure of the virus particles. Hence, the AFM approach provides a starting point for further detailed studies on TSWV ribonucleoprotein complexes.     

Self assembly of epicuticular waxes on living plant surfaces imaged by atomic force microscopy (AFM)

The cuticle of terrestrial vascular plants and some bryophytes is covered with a complex mixture of lipids, usually called epicuticular waxes. Self-assembly processes of wax molecules lead to crystalline three-dimensional micro- and nanostructures that emerge from an underlying wax film. This paper presents the first AFM study on wax regeneration on the surfaces of living plants and the very early stages of wax crystal formation at the molecular level. Wax formation was analysed on the leaves of Euphorbia lathyris, Galanthus nivalis, and Ipheion uniflorum. Immediately after wax removal, regeneration of a wax film began, consisting of individual layers of, typically, 3-5 nm thickness. Subsequently, several different stages of crystal growth could be distinguished, and different patterns of wax regeneration as well as considerable variation in regeneration speed were found.     

Solid-state microstructure of poly(l-lactide) and l-lactide/meso-lactide random copolymers by atomic force microscopy (AFM)

Tapping mode atomic force microscopy was used to investigate the lamellar morphology of poly(l-lactide) and two poly(l-lactide-co-meso-lactide) random copolymers containing 3% and 6% meso-lactide. Samples were isothermally crystallized at selected temperatures, and qualitative and quantitative analyses of lamellar structure were performed using height and phase images. This is the first study of the morphology of polylactide stereocopolymers using a real-space probe, and the important effects of scanning parameters on the acquired images are described. More open spherulites with an abundance of screw dislocations between edge-on lamellar stacks were observed in samples crystallized at higher temperatures. Mean lamellar thicknesses are lower for the random copolymers compared to PLLA, particularly at lower DeltaT, in agreement with the results of our previous small-angle X-ray scattering (SAXS) experiments. Mean lamellar thicknesses derived from the current real-space examination are in good agreement with those determined previously from SAXS. Internal surfaces-from microtomed specimens-were also studied to investigate the bulk crystal morphology. Although quantitative analysis was not feasible (for reasons discussed in the text), lamellar organization similar to that seen in the surface experiments is observed at high magnifications.     

DNA binding domain in the replication checkpoint protein Mrc1 of Schizosaccharomyces pombe

The replication checkpoint is activated when replication forks are obstructed by DNA lesions or protein complexes bound to DNA or when DNA synthesis is restrained by the limited availability of deoxyribonucleotides. This checkpoint preserves genome integrity by stabilizing stalled forks and delaying the onset of mitosis. In the fission yeast Schizosaccharomyces pombe, Mrc1 is a replication checkpoint adaptor protein that allows the sensor kinase Rad3-Rad26 to activate the effector kinase Cds1. In Saccharomyces cerevisiae, Mrc1 associates with replication forks and co-precipitates with the DNA replication protein Cdc45. Whether or not Mrc1 interacts directly with DNA is unknown. Here we define a approximately 150 amino acid DNA binding domain (DBD) in the N-terminal region of S. pombe Mrc1. The DBD interacts preferentially with branched DNA structures in vitro. Deletion of the DBD or point mutations that diminish its DNA binding activity render cells sensitive to the replication inhibitor hydroxyurea. These mutations also impair the replication checkpoint arrest. The DBD has a helix-loop-helix motif that is predicted to bind DNA. This motif is conserved in the recently identified N-terminal DBD of human Claspin, a presumptive homolog of yeast Mrc1 proteins.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr³⁺ ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr³⁺ ion binding mainly to N₇ (G) and to phosphate groups was demonstrated. ψ‐Type VCD spectra resembling electronic CD (ECD) spectra, which appear during ψ‐type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large‐scale helicity. Atomic force microscopy confirmed DNA condensation by Cr³⁺ ions and the formation of tight DNA particles responsible for the ψ‐type VCD spectra. Upon increasing the Cr³⁺ ion concentration the shape of the condensates changed from loose flower‐like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr³⁺ ions studied. The secondary structure of DNA remained in a B‐form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles. © 2002 Wiley Periodicals, Inc. Biopolymers 61: 243–260, 2002     

Rapid self-assembly of alpha-synuclein observed by in situ atomic force microscopy

Self-assembly of alpha-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type alpha-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant alpha-synuclein(1-108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(+/-0.2)nm in height and presumably representing individual beta-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote alpha-synuclein self-assembly. In the presence of spermidine or spermine 5.9(+/-1.0)nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. alpha-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on alpha-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of alpha-synuclein self-assembly and identifies key factors modulating the aggregation process.     

Vibrational CD (VCD) and atomic force microscopy (AFM) study of DNA interaction with Cr3+ ions: VCD and AFM evidence of DNA condensation

The interaction of natural calf thymus DNA with Cr(3+) ions was studied at room temperature by means of vibrational CD (VCD) and infrared absorption (ir) spectroscopy, and atomic force microscopy (AFM). Cr(3+) ion binding mainly to N(7) (G) and to phosphate groups was demonstrated. Psi-type VCD spectra resembling electronic CD (ECD) spectra, which appear during psi-type DNA condensation, were observed. These spectra are characterized mainly by an anomalous, severalfold increase of VCD intensity. Such anomalous VCD spectra were assigned to DNA condensation with formation of large and dense particles of a size comparable to the wavelength of the probing ir beam and possessing large-scale helicity. Atomic force microscopy confirmed DNA condensation by Cr(3+) ions and the formation of tight DNA particles responsible for the psi-type VCD spectra. Upon increasing the Cr(3+) ion concentration the shape of the condensates changed from loose flower-like structures to highly packed dense spheres. No DNA denaturation was seen even at the highest concentration of Cr(3+) ions studied. The secondary structure of DNA remained in a B-form before and after the condensation. VCD and ir as well as AFM proved to be an effective combination for investigating DNA condensation. In addition to the ability of VCD to determine DNA condensation, VCD and ir can in the same experiment provide unambiguous information about the secondary structure of DNA contained in the condensed particles.     

Isolation, Characterization, and Molecular Cloning of a Protein (Abp2) That Binds to a Schizosaccharomyces pombe Origin of Replication (ars3002)

The autonomously replicating sequence (ARS) element ars3002 is associated with the most active replication origin within a cluster of three closely spaced origins on chromosome III of Schizosaccharomyces pombe. A 361-bp portion of ars3002 containing detectable ARS activity includes multiple near matches to the S. pombe ARS consensus sequence previously reported by Maundrell et al. (K. Maundrell, A. Hutchison, and S. Shall, EMBO J. 7:2203–2209, 1988). Using a gel shift assay with a multimer of an oligonucleotide containing three overlapping matches to the Maundrell ARS consensus sequence, we have detected several proteins in S. pombe crude extracts that bind to the oligonucleotide and ars3002. One of these proteins, ARS binding protein 1, was previously described (Abp1 [Y. Murakami, J. A. Huberman, and J. Hurwitz, Proc. Natl. Acad. Sci. USA 93:502–507, 1996]). In this report the isolation, characterization, and cloning of a second binding activity, designated ARS binding protein 2 (Abp2), are described. Purified Abp2 has an apparent molecular mass of 75 kDa. Footprinting analyses revealed that it binds preferentially to overlapping near matches to the Maundrell ARS consensus sequence. The gene abp2 was isolated, sequenced, and overexpressed in Escherichia coli. The DNA binding activity of overexpressed Abp2 was similar to that of native Abp2. The deduced amino acid sequence contains a region similar to a proline-rich motif (GRP) present in several proteins that bind A+T-rich DNA sequences. Replacement of amino acids within this motif with alanine either abolished or markedly reduced the DNA binding activity of the mutated Abp2 protein, indicating that this motif is essential for the DNA binding activity of Abp2. Disruption of the abp2 gene showed that the gene is not essential for cell viability. However, at elevated temperatures the null mutant was less viable than the wild type and exhibited changes in nuclear morphology. The null mutant entered mitosis with delayed kinetics when DNA replication was blocked with hydroxyurea, and advancement through mitosis led to the loss of cell viability and aberrant formation of septa. The null mutant was also sensitive to UV radiation, suggesting that Abp2 may play a role in regulating the cell cycle response to stress signals.