Mechanism Of Disassembly Research Articles

Quantitative analysis of human keratinocyte cell elasticity using atomic force microscopy (AFM).

We present the use of atomic force microscopy (AFM) to visualize and quantify the dynamics of epithelial cell junction interactions under physiological and pathophysiological conditions at the nanoscale. Desmosomal junctions are critical cellular adhesion components within epithelial tissues and blistering skin diseases such as Pemphigus are the result in the disruption of these components. However, these structures are complex and mechanically inhomogeneous, making them difficult to study. The mechanisms of autoantibody mediated keratinocyte disassembly remain largely unknown. Here, we have used AFM technology to image and measure the mechanical properties of living skin epithelial cells in culture. We demonstrate that force measurement data can distinguish cells cultured with and without autoantibody treatment. Our demonstration of the use of AFM for in situ imaging and elasticity measurements at the local, or tissue level opens potential new avenues for the investigation of disease mechanisms and monitoring of therapeutic strategies in blistering skin diseases.

Single-molecule analysis of a molecular disassemblase reveals the mechanism of Hsc70-driven clathrin uncoating

Heat shock cognate protein 70 (Hsc70) supports remodeling of protein complexes -- for example, disassembly of clathrin coats on endocytic coated vesicles. To understand how a simple ATP driven molecular clamp catalyzes a large-scale disassembly reaction, we have used single-particle fluorescence imaging to track the dynamics of Hsc70 and its clathrin substrate in real time. Hsc70 accumulates to a critical level, determined by kinetic modeling to be one Hsc70 for every two functional attachment sites; rapid, all-or-none uncoating then ensues. We propose that Hsc70 traps conformational distortions, seen previously by electron cryomicroscopy, in the vicinity of each occupied site and that accumulation of local strains destabilises the clathrin lattice. Capture of conformational fluctuations may be a general mechanism for chaperone-driven disassembly of protein complexes.

9J-02 アクチンストレスファイバが張力依存的に脱重合するメカニズムの考察(OS-11(3) 細胞・生体分子のバイオメカニクス(1))

9J-02 アクチンストレスファイバが張力依存的に脱重合するメカニズムの考察(OS-11(3) 細胞・生体分子のバイオメカニクス(1))

Kinesins at a glance.

Kinesin was discovered in 1985 – 25 years ago – based on its motility in cytoplasm extruded from the giant axon of the squid ([Allen et al., 1982][1]; [Brady et al., 1982][2]; [Vale et al., 1985][3]). The purified protein, now known as kinesin-1, consisted of two heavy chains with motor activity

Assembly/Disassembly of Drug Conjugates Using Imide Ligation

A strategy is described that allows the easy assembly and controlled disassembly of drug conjugates. Imide ligation, that is, the reaction of a peptide thioacid with an azidoformate, is used for conjugate assembly. The imide bond participates also with an endopeptidase-triggered cyclization-based disassembly mechanism.

Unconventional Processive Mechanics of Non-muscle Myosin IIB

Proper tension maintenance in the cytoskeleton is essential for regulated cell polarity, cell motility, and division. Non-muscle myosin IIB (NMIIB) generates tension along actin filaments in many cell types, including neuronal, cardiac, and smooth muscle cells. Using a three-bead optical trapping assay, we recorded NMIIB interactions with actin filaments to determine if a NMIIB dimer cycles along an actin filament in a processive manner. Our results show that NMIIB is the first myosin II to exhibit evidence of processive stepping behavior. Analysis of these data reveals a forward displacement of 5.4 nm and, surprisingly, frequent backward steps of -5.9 nm. Processive stepping along the long pitch helix of actin may provide a mechanism for disassembly of fascin-actin bundles. Forward steps and detachment are weakly force-dependent at all forces, consistent with rate-limiting and force-dependent ADP release. However, backward steps are nearly force-independent. Our data support a model in which NMIIB can readily move in both directions at stall, which may be important for a general regulator of cytoskeleton tension.

Quantitative Analysis of the Mechanism of Endocytic Actin Patch Assembly and Disassembly in Fission Yeast

We used quantitative confocal microscopy to measure the numbers of 16 proteins tagged with fluorescent proteins during assembly and disassembly of endocytic actin patches in fission yeast. The peak numbers of each molecule that accumulate in patches varied <30-50% between individual patches. The pathway begins with accumulation of 30-40 clathrin molecules, sufficient to build a hemisphere at the tip of a plasma membrane invagination. Thereafter precisely timed waves of proteins reach characteristic peak numbers: endocytic adaptor proteins (approximately 120 End4p and approximately 230 Pan1p), activators of Arp2/3 complex (approximately 200 Wsp1p and approximately 340 Myo1p) and approximately 300 Arp2/3 complexes just ahead of a burst of actin assembly into short, capped and highly cross-linked filaments (approximately 7000 actins, approximately 200 capping proteins, and approximately 900 fimbrins). Coronin arrives last as all other components disperse upon patch internalization and movement over approximately 10 s. Patch internalization occurs without recruitment of dynamins. Mathematical modeling, described in the accompanying paper (Berro et al., 2010, MBoC 21: 2803-2813), shows that the dendritic nucleation hypothesis can account for the time course of actin assembly into a branched network of several hundred filaments 100-200 nm long and that patch disassembly requires actin filament fragmentation in addition to depolymerization from the ends.

Insight into the Mechanisms of Adenovirus Capsid Disassembly from Studies of Defensin Neutralization

Defensins are effectors of the innate immune response with potent antibacterial activity. Their role in antiviral immunity, particularly for non-enveloped viruses, is poorly understood. We recently found that human alpha-defensins inhibit human adenovirus (HAdV) by preventing virus uncoating and release of the endosomalytic protein VI during cell entry. Consequently, AdV remains trapped in the endosomal/lysosomal pathway rather than trafficking to the nucleus. To gain insight into the mechanism of defensin-mediated neutralization, we analyzed the specificity of the AdV-defensin interaction. Sensitivity to alpha-defensin neutralization is a common feature of HAdV species A, B1, B2, C, and E, whereas species D and F are resistant. Thousands of defensin molecules bind with low micromolar affinity to a sensitive serotype, but only a low level of binding is observed to resistant serotypes. Neutralization is dependent upon a correctly folded defensin molecule, suggesting that specific molecular interactions occur with the virion. CryoEM structural studies and protein sequence analysis led to a hypothesis that neutralization determinants are located in a region spanning the fiber and penton base proteins. This model was supported by infectivity studies using virus chimeras comprised of capsid proteins from sensitive and resistant serotypes. These findings suggest a mechanism in which defensin binding to critical sites on the AdV capsid prevents vertex removal and thereby blocks subsequent steps in uncoating that are required for release of protein VI and endosomalysis during infection. In addition to informing the mechanism of defensin-mediated neutralization of a non-enveloped virus, these studies provide insight into the mechanism of AdV uncoating and suggest new strategies to disrupt this process and inhibit infection.

Actin stress fibers are at a tipping point between conventional shortening and rapid disassembly at physiological levels of MgATP

Stress fibers (SFs) composed of nonmuscle actin and myosin II play critical roles in various cellular functions such as structural remodeling in response to changes in cell stress or strain. Previous studies report that SFs rapidly disassemble upon loss of tension caused by reduced myosin activity or sudden cell shortening, but the mechanism is unclear. Here, we showed that Rho-kinase inhibition with Y-27632 led to detachment of intact actin filaments from the SFs rather than depolymerization. Loss of tension may allow SFs to shorten via MgATP-driven cross-bridge cycling, thus we investigated the effects of MgATP concentration on SF shortening and stability. We performed the experiments using extracted SFs to allow control over MgATP concentration. SF contraction and disassembly rates each increased with increasing MgATP concentration. SFs transitioned from conventional SF shortening to rapid disassembly as MgATP concentration increases from 2 to 5 mM, which is within the physiological range of intracellular MgATP concentrations. Thus, we submit that SFs in intact cells are inherently on the verge of disassembly, which is likely due to the small number of actomyosin cross-bridges in SFs compared to those found in relatively stable myofibrils. Given that recent studies have revealed that loss of resistive force against myosin II could lower the fraction of the MgATPase cycle time that the myosin head is attached to actin (i.e., the duty ratio), binding of cytoplasmic levels of MgATP to myosin II may be sufficient to cause the disassembly of unloaded SFs. The present study thus describes a putative mechanism for rapid SF disassembly caused by decreased myosin activity or sudden cell shortening.

Tracking the dynamic interplay between bacterial and host factors during pathogen-induced vacuole rupture in real time

Escape into the host cell cytosol following invasion of mammalian cells is a common strategy used by invasive pathogens. This requires membrane rupture of the vesicular or vacuolar compartment formed around the bacteria after uptake into the host cell. The mechanism of pathogen-induced disassembly of the vacuolar membrane is poorly understood. We established a novel, robust and sensitive fluorescence microscopy method that tracks the precise time point of vacuole rupture upon uptake of Gram-negative bacteria. This revealed that the enteroinvasive pathogen Shigella flexneri escapes rapidly, in less than 10 min, from the vacuole. Our method demonstrated the recruitment of host factors, such as RhoA, to the bacterial entry site and their continued presence at the point of vacuole rupture. We found a novel host marker for ruptured vacuoles, galectin-3, which appears instantly in the proximity of bacteria after escape into the cytosol. Furthermore, we show that the Salmonella effector proteins, SifA and PipB2, stabilize the vacuole membrane inhibiting bacterial escape from the vacuole. Our novel approach to track vacuole rupture is ideally suited for high-content and high-throughput approaches to identify the molecular and cellular mechanisms of membrane rupture during invasion by pathogens such as viruses, bacteria and parasites.

Crystal Structures of P. Aeruginosa Reveal a Dynamic type IV Pilus Motor Protein

Type IV pili are extracellular bacterial virulence factors that are retracted into cells by the powerful molecular motor, PilT. Pseudomonas aeruginosa PilT crystal structures, both AMP-PCP bound and unliganded, have been refined at 2.6 and 3.1 Å resolution, respectively. The structures reveal an interlocking asymmetric hexamer mortared with extensive ionic interactions. The three subunits in each asymmetric unit exhibit differing conformations, implying domain motions during the ATP-coupled mechanism of pilus retraction and disassembly into membrane-localized pilin monomers. The force-generating swing of PilT upon nucleotide binding has a magnitude of ∼20° and a direction diagonal to the polar axis. Future work will focus on identifying protein interaction partners of PilT to more fully understand the pilus retraction process.

Counting RAD51 Proteins Disassembling from Nucleoprotein Filaments Under Tension

The central catalyst in eukaryotic ATP-dependent homologous recombination consists of RAD51 proteins, polymerized around single-stranded DNA. This nucleoprotein filament recognizes a homologous duplex DNA segment and invades it. After strand exchange, the nucleoprotein filament should disassemble in order for the recombination process to complete. The molecular mechanism of RAD51 filament disassembly is poorly understood. Here, we have combined optical tweezers with single-molecule fluorescence microscopy and microfluidics to reveal that disassembly results from the interplay between ATP hydrolysis and release of the tension stored in the nucleoprotein filament [1]. Applying external tension to the DNA, we found that disassembly slows down and can even be stalled. We quantified the fluorescence of RAD51 patches and found that disassembly occurs in bursts interspersed by long pauses. Upon relaxation of a stalled complex, pauses were suppressed resulting in a large burst. These results imply that tension-dependent disassembly takes place only from filament ends, after tension-independent ATP hydrolysis. This integrative single-molecule approach allowed us to dissect the mechanism of this key homologous recombination reaction step, which in turn clarifies how disassembly can be influenced by accessory proteins. [1] van Mameren et al., Nature, 457, 745 - 748 (2009)

The stability of imine-containing dynamic [2]rotaxanes to hydrolysis

Large amounts (>100 mol equivalents) of water are required to effect by hydrolysis the partial disassembly of the rings from the dumbbell components of two dynamic [2]rotaxanes. The two dynamic [2]rotaxanes are comprised of [24]crown-8 rings-each of which incorporate two imine bonds-encircling a dumbbell component composed of a dibenzylammonium ion in which each of the two benzyl substituents carries two methoxyl groups attached to their 3- and 5-positions. A mechanism for the partial disassembly of the two dynamic [2]rotaxanes, involving the cleavage of the kinetically labile imine bonds by water molecules, is proposed. The most important experimental observation to be noted is the fact that the hydrolysis of the macrocyclic diimines, associated with the templating -CH(2)NH(2)(+)CH(2)-centres in the middle of their dumbbells, turns out to be an uphill task to perform in the face of the molecular recognition provided by strong [N(+)-HO] hydrogen bonds and weaker, yet not insignificant, [C-HO] interactions. The dynamic nature of the imine bond formation and hydrolysis is such that the acyclic components produced during hydrolysis of the imine bonds can be enticed to cyclise once again around the -CH(2)NH(2)(+)CH(2)-template, affording the [2]rotaxanes. The reluctance of imine bonds, present in substantial numbers in larger molecular and extended structures, is significant when it comes to exercising dynamic chemistry in compounds where multiple imine bonds are present.

Coupling DNA Unwinding Activity With Primer Synthesis in the Bacteriophage T4 Primosome

The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp4) translocating 5′ to 3′ and an oligomeric primase (gp6) synthesizing primers 5′ to 3′, have been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a new DNA looping mechanism. A fused gp6-gp4primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms, which disfavor primosome disassembly, thereby increasing primase processivity. In contrast, priming in bacteriophage T7 involves discrete pausing of the primosome, and in Escherichia coli it appears to be associated primarily with dissociation of the primase from the helicase. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.

Structure of clathrin coat with bound Hsc70 and auxilin: mechanism of Hsc70-facilitated disassembly

The chaperone Hsc70 drives the clathrin assembly–disassembly cycle forward by stimulating dissociation of a clathrin lattice. A J-domain containing co-chaperone, auxilin, associates with a freshly budded clathrin-coated vesicle, or with an in vitro assembled clathrin coat, and recruits Hsc70 to its specific heavy-chain-binding site. We have determined by electron cryomicroscopy (cryoEM), at about 11 Å resolution, the structure of a clathrin coat (in the D6-barrel form) with specifically bound Hsc70 and auxilin. The Hsc70 binds a previously analysed site near the C-terminus of the heavy chain, with a stoichiometry of about one per three-fold vertex. Its binding is accompanied by a distortion of the clathrin lattice, detected by a change in the axial ratio of the D6 barrel. We propose that when Hsc70, recruited to a position close to its target by the auxilin J-domain, splits ATP, it clamps firmly onto its heavy-chain site and locks in place a transient fluctuation. Accumulation of the local strain thus imposed at multiple vertices can then lead to disassembly.

Coupling DNA unwinding activity with primer synthesis in the bacteriophage T4 primosome

The unwinding and priming activities of the bacteriophage T4 primosome, which consists of a hexameric helicase (gp41) translocating 5′ to 3′ and an oligomeric primase (gp61) synthesizing primers 5′ to 3′, has been investigated on DNA hairpins manipulated by a magnetic trap. We find that the T4 primosome continuously unwinds the DNA duplex while allowing for primer synthesis through a primosome disassembly mechanism or a novel DNA looping mechanism. A fused gp61-gp41 primosome unwinds and primes DNA exclusively via the DNA looping mechanism. Other proteins within the replisome control the partitioning of these two mechanisms disfavoring primosome disassembly thereby increasing primase processivity. In contrast priming in bacteriophage T7 involves discrete pausing of the primosome and in Escherichia coli appears to be associated primarily with dissociation of the primase from the helicase. Thus nature appears to use several strategies to couple the disparate helicase and primase activities within primosomes.

Structural Plasticity in Actin and Tubulin Polymer Dynamics

Actin filaments and microtubules polymerize and depolymerize by adding and removing subunits at polymer ends, and these dynamics drive cytoplasmic organization, cell division, and cell motility. Since Wegner proposed the treadmilling theory for actin in 1976, it has largely been assumed that the chemical state of the bound nucleotide determines the rates of subunit addition and removal. This chemical kinetics view is difficult to reconcile with observations revealing multiple structural states of the polymer that influence polymerization dynamics but that are not strictly coupled to the bound nucleotide state. We refer to these phenomena as "structural plasticity" and discuss emerging evidence that they play a central role in polymer dynamics and function.

FACT and the Proteasome Promote Promoter Chromatin Disassembly and Transcriptional Initiation

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.

Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR.

Redox-regulated Export of the Major Histocompatibility Complex Class I-Peptide Complexes from the Endoplasmic Reticulum

In contrast to the fairly well-characterized mechanism of assembly of MHC class I-peptide complexes, the disassembly mechanism by which peptide-loaded MHC class I molecules are released from the peptide-loading complex and exit the endoplasmic reticulum (ER) is poorly understood. Optimal peptide binding by MHC class I molecules is assumed to be sufficient for triggering exit of peptide-filled MHC class I molecules from the ER. We now show that protein disulfide isomerase (PDI) controls MHC class I disassembly by regulating dissociation of the tapasin-ERp57 disulfide conjugate. PDI acts as a peptide-dependent molecular switch; in the peptide-bound state, it binds to tapasin and ERp57 and induces dissociation of the tapasin-ERp57 conjugate. In the peptide-free state, PDI is incompetent to bind to tapasin or ERp57 and fails to dissociate the tapasin-ERp57 conjugates, resulting in ER retention of MHC class I molecules. Thus, our results indicate that even after optimal peptide loading, MHC class I disassembly does not occur by default but, rather, is a regulated process involving PDI-mediated interactions within the peptide-loading complex.