Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

University of Utah

Under sustained pumping, kinetics of macroscopic nonlinear biochemical reaction systems far from equilibrium either can be in a stationary steady state or can execute sustained oscillations about a fixed mean. For a system of two dynamic species X and Y, the concentrations n(x) and n(y) will be constant or will repetitively trace a closed loop in the (n(x), n(y)) phase plane, respectively. We study a mesoscopic system with n(x) and n(y) very small; hence the occurrence of random fluctuations modifies the deterministic behavior and the law of mass action is replaced by a stochastic model. We show that n(x) and n(y) execute cyclic random walks in the (n(x), n(y)) plane whether or not the deterministic kinetics for the corresponding macroscopic system represents a steady or an oscillating state. Probability distributions and correlation functions for n(x)(t) and n(y)(t) show quantitative but not qualitative differences between states that would appear as either oscillating or steady in the corresponding macroscopic systems. A diffusion-like equation for probability P(n(x), n(y), t) is obtained for the two-dimensional Brownian motion in the (n(x), n(y)) phase plane. In the limit of large n(x), n(y), the deterministic nonlinear kinetics derived from mass action is recovered. The nature of large fluctuations in an oscillating nonequilibrium system and the conceptual difference between "thermal stochasticity" and "temporal complexity" are clarified by this analysis. This result is relevant to fluorescence correlation spectroscopy and metabolic reaction networks.

Concentration fluctuations in a mesoscopic oscillating chemical reaction system.

Fluorescence correlation spectroscopy (FCS) measurements have been carried out on the intestinal fatty acid binding protein (IFABP) to study microsecond dynamics of the protein in its native state as well as in pH-induced intermediates. IFABP is a small (15 kDa) protein that consists mostly of antiparallel beta-strands enclosing a large central cavity into which the ligand binds. Because this protein does not contain cysteine, two cysteine mutants (Val60Cys and Phe62Cys) have been prepared and covalently modified with fluorescein. Based on fluorescence measurements, one of the mutants (Val60Flu) has the fluorescein moiety inside the cavity of the protein, whereas the fluorescein is exposed to solvent in the other (Phe62Flu). The protein modified at position 60 demonstrates the presence of a conformational event on the order of 35 microsec, which is not seen in the other mutant (Phe62Flu). The amplitude of this fast conformational event decreases sharply at low pH as the protein unfolds. Experiments measuring the diffusion as a function of pH indicate the formation of a compact state distinct from the native state at about pH 3.5. Steady state fluorescence and far-UV CD indicates that unfolding occurs at pH values below pH 3.

Measurement of microsecond dynamic motion in the intestinal fatty acid binding protein by using fluorescence correlation spectroscopy.

We present a detailed statistical analysis of fluorescence correlation spectroscopy for a wide range of timescales. The derivation is completely analytical and can provide an excellent tool for planning and analysis of FCS experiments. The dependence of the signal-to-noise ratio on different measurement conditions is extensively studied. We find that in addition to the shot noise and the noise associated with correlated molecular dynamics there is another source of noise that appears at very large lag times. We call this the "particle noise," as its behavior is governed by the number of particles that have entered and left the laser beam sample volume during large dwell times. The standard deviations of all the points on the correlation function are calculated analytically and shown to be in good agreement with experiments. We have also investigated the bias associated with experimental correlation function measurements. A "phase diagram" for FCS experiments is constructed that demonstrates the significance of the bias for any given experiment. We demonstrate that the value of the bias can be calculated and added back as a first-order correction to the experimental correlation function.

Statistical analysis of fluorescence correlation spectroscopy: the standard deviation and bias.

We show that activated collagenase (MMP-1) moves processively on the collagen fibril. The mechanism of movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. Inactivation of the enzyme by a single amino acid residue substitution in the active center eliminates the bias without noticeable effect on rate of diffusion. Monte Carlo simulations using a model similar to a "burnt bridge" Brownian ratchet accurately describe our experimental results and previous observations on kinetics of collagen digestion. The biological implications of MMP-1 acting as a molecular ratchet tethered to the cell surface suggest new mechanisms for its role in tissue remodeling and cell-matrix interaction.

Interstitial collagenase is a Brownian ratchet driven by proteolysis of collagen.

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil.

Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy.

Biased diffusion of collagenase on collagen fibrils may represent the first observed adenosine triphosphate-independent extracellular molecular motor. The magnitude of force generated by the enzyme remains unclear. We propose a propulsion mechanism based on a burnt bridges Brownian ratchet model with a varying degree of coupling of the free energy from collagen proteolysis to the enzyme motion. When constrained by experimental observations, our model predicts 0.1 pN stall force for individual collagenase molecules. A dimer, surprisingly, can generate a force in the range of 5 pN, suggesting that the motor can be of biological significance.

Powering a burnt bridges Brownian ratchet: a model for an extracellular motor driven by proteolysis of collagen.

&#x7F51;&#x683C;&#x86CB;&#x767D;&#x6D82;&#x5C42;&#x5751;&#x548C;&#x5176;&#x88AB;&#x819C;&#x5C0F;&#x6CE1;&#x6210;&#x7684;&#x6210;&#x719F;&#x7EC4;&#x4EF6;&#xFF0C;&#x9700;&#x8981;&#x534F;&#x8C03;&#x5177;&#x4F53;&#x8840;&#x8102;&#x548C;&#x51E0;&#x4E2A;&#x7ED3;&#x6784;&#x548C;&#x89C4;&#x7BA1;&#x86CB;&#x767D;&#x8D28;&#x4E4B;&#x95F4;&#x7684;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x3002;&#x4E0B;&#x4E3B;&#x9187;&#x3001; &#x78F7;&#x8102;&#x9176; D &#x751F;&#x6210; phosphatidylalcohols&#xFF0C;&#x800C;&#x4E0D;&#x662F;&#x5DF4;&#x52D2;&#x65AF;&#x5766;&#x6743;&#x529B;&#x673A;&#x6784;&#xFF0C;&#x51CF;&#x5C11;&#x523A;&#x6FC0;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187; 5-&#x6FC0;&#x9176; (PI5K) &#x5E76;&#x56E0;&#x6B64;&#x964D;&#x4F4E;&#x5F62;&#x6210;&#x7684;&#x78F7;&#x8102;&#x9170;&#x808C;&#x9187;-4&#xFF0C;5-&#x6C22;&#x9499; &#xFF08;PIP(2))&#x3002;&#x7528;&#x6D3B;&#x7EC6;&#x80DE;&#x6210;&#x50CF;&#xFF0C;&#x6211;&#x4EEC;&#x5DF2;&#x8868;&#x660E; 1-&#x4E01;&#x9187;&#x6216;&#x5176;&#x4ED6;&#x5C0F;&#x4E3B;&#x9187;&#x4E0E;&#x7EC6;&#x80DE;&#x6CBB;&#x7597;&#x6025;&#x6027;&#x8BF1;&#x5BFC;&#x5FEB;&#x901F;&#x62C6;&#x5378;&#x7684;&#x7B49;&#x79BB;&#x5B50;&#x819C;&#x6D82;&#x5C42;&#x5751;&#xFF0C;&#x5E76;&#x963B;&#x6B62;&#x65B0;&#x7684;&#x5916;&#x89C2;&#x3002;&#x5916;&#x6E90; PIP(2) &#x53E6;&#x5916;&#x53CD;&#x8F6C;&#x6B64;&#x6548;&#x679C;&#x3002;&#x91CD;&#x65B0;&#x6D82;&#x7684;&#x5751;&#x53CA;&#x6C34;&#x6CE1;&#x51FA;&#x73B0;&#x540C;&#x6B65; 1-&#x4E01;&#x9187; &#xFF1B; &#x53D6;&#x6D88;&#x540E;&#x6211;&#x4EEC;&#x4F7F;&#x7528;&#x6B64;&#x540C;&#x6B65;&#x6027;&#x8BC4;&#x4F30;&#x9540;&#x819C;&#x7684;&#x56CA;&#x6CE1;&#x5927;&#x4F1A;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x7684;&#x4F5C;&#x7528;&#x3002;&#x957F;&#x65F6;&#x95F4;&#x7684;&#x6291;&#x5236;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x805A;&#x5408;&#x7531; latrunculin A &#x6216; cytochalasin D &#x51CF;&#x5C11;&#x5927;&#x7EA6; 50%&#x6D82;&#x7684;&#x5751;&#x5F62;&#x6210;&#x7684;&#x9891;&#x7387;&#x800C;&#x4E0D;&#x4F1A;&#x5F71;&#x54CD;&#x5230;&#x88AB;&#x819C;&#x5C0F;&#x6CE1;&#x7684;&#x6210;&#x719F;&#x3002;&#x5728;&#x63A7;&#x5236;&#x7EC6;&#x80DE;&#x53BB;&#x9664; 1-&#x4E01;&#x9187;&#x4E2D;&#x7684;&#x808C;&#x52A8;&#x86CB;&#x767D; depolymerizer &#x7EE7;&#x7EED;&#x5B58;&#x5728;&#x5BFC;&#x81F4;&#x540C;&#x6B65;&#x7684;&#x65B0;&#x5751;&#xFF0C;&#x4E00;&#x822C;&#x6210;&#x719F;&#x7684;&#x5916;&#x89C2;&#x3002;&#x56E0;&#x6B64;&#xFF0C;&#x91CD;&#x5851;&#x7684;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x7EC6;&#x80DE;&#x9AA8;&#x67B6;&#x662F;&#x6CA1;&#x6709;&#x5FC5;&#x8981;&#x6D82;&#x8986;&#x7F51;&#x683C;&#x86CB;&#x767D;&#x56CA;&#x6CE1;&#x5927;&#x4F1A;&#x4F46;&#x53EF;&#x80FD;&#x95F4;&#x63A5;&#x5F71;&#x54CD;&#x7684;&#x7F51;&#x683C;&#x86CB;&#x767D;&#x6D82;&#x5C42;&#x5751;&#x6210;&#x6838;&#x3002;

&#x8840;&#x8102;&#x548C;&#x808C;&#x52A8;&#x86CB;&#x767D;&#x4E2D;&#x7684;&#x7F51;&#x683C;&#x86CB;&#x767D;&#x6D82;&#x5C42;&#x5751;&#x5F62;&#x6210;&#x7684;&#x4F5C;&#x7528;&#x3002;

Assembly of clathrin-coated pits and their maturation into coated vesicles requires coordinated interactions between specific lipids and several structural and regulatory proteins. In the presence of primary alcohols, phospholipase D generates phosphatidylalcohols instead of PA, reducing stimulation of phosphatidyl inositol 5-kinase (PI5K) and hence decreasing formation of phosphoinositide-4,5-biphosphate (PIP(2)). Using live-cell imaging, we have shown that acute treatment of cells with 1-butanol or other small primary alcohols induces rapid disassembly of coated pits at the plasma membrane and blocks appearance of new ones. Addition of exogenous PIP(2) reverses this effect. Coated pits and vesicles reappear synchronously upon removal of 1-butanol; we have used this synchrony to assess the role of actin in coated vesicle assembly. Prolonged inhibition of actin polymerization by latrunculin A or cytochalasin D reduced by approximately 50% the frequency of coated pit formation without affecting maturation into coated vesicles. As in control cells, removal of 1-butanol in the continued presence of an actin depolymerizer led to synchronous appearance of new pits, which matured normally. Thus, remodeling of the actin cytoskeleton is not essential for clathrin-coated vesicle assembly but may indirectly affect the nucleation of clathrin-coated pits.

Role of lipids and actin in the formation of clathrin-coated pits.

Ensemble des fosses recouvertes de clathrine et leur maturation dans des v&eacute;sicules enduits n&eacute;cessite une coordonn&eacute;e des interactions entre lipides sp&eacute;cifiques et plusieurs prot&eacute;ines structurelles et r&eacute;glementaires. En pr&eacute;sence d&#39;alcools primaires, phospholipase D g&eacute;n&egrave;re phosphatidylalcohols au lieu de PA, r&eacute;duire la stimulation de la phosphatidyl inositol 5-kinase (PI5K) et donc diminue la formation des phosphoinositides-4, 5-biphosphate (PIP(2)). &Agrave; l&#39;aide d&#39;imagerie de cellules vivantes, nous avons montr&eacute; qu&#39;un traitement aigu des cellules avec le butan-1-ol ou d&#39;autres alcools primaires petits induit un d&eacute;montage rapid des puits recouverts &agrave; la membrane plasmique et bloque l&#39;apparition de nouvelles. Ajout de PIP(2) exog&egrave;ne inverse &agrave; cet effet. V&eacute;sicules et puits recouverts r&eacute;appara&icirc;tront de fa&ccedil;on synchrone lors du retrait du butan-1-ol ; Nous avons utilis&eacute; cette synchronie afin d&#39;&eacute;valuer le r&ocirc;le de l&#39;actine en Assembl&eacute;e v&eacute;sicule couch&eacute;. Inhibition prolong&eacute;e de la polym&eacute;risation de l&#39;actine par Latrunculine A ou cytochalasine D r&eacute;duit d&#39;environ 50 % la fr&eacute;quence de la formation de puits tapiss&eacute;s sans affecter la maturation dans les v&eacute;sicules de l&#39;enduit. Comme dans les cellules t&eacute;moins, enl&egrave;vement de butan-1-ol dans la pr&eacute;sence d&#39;un depolymerizer d&#39;actine conduit &agrave; synchrone apparition de nouveaux noyaux, qui normalement est arriv&eacute;e &agrave; &eacute;ch&eacute;ance. Ainsi, le remodelage du cytosquelette d&#39;actine n&#39;est pas indispensable pour l&#39;assemblage de clathrin-coated vesicle mais peut influer indirectement sur la nucl&eacute;ation des fosses recouvertes de clathrine.

R&ocirc;le des lipides et l&#39;actine dans la formation de fosses recouvertes de clathrine.

Montage von Clathrin-coated Pits und ihre Reifung in beschichteten Vesikel erfordert koordinierte Wechselwirkungen zwischen bestimmten Lipiden und mehreren strukturellen und regulatorischen Proteinen. In Anwesenheit von prim&auml;ren Alkoholen generiert die Phospholipase D Phosphatidylalcohols anstelle von PA, Verringerung der Stimulation des phosphatidyles Inosit 5-Kinase (PI5K) und somit abnehmender Bildung von Phosphoinositid-4,5-Biphosphate (PIP(2)). Leben-Zelle Darstellung verwenden, haben wir gezeigt, dass akute Behandlung der Zellen mit 1-Butanol oder anderen kleinen prim&auml;ren Alkoholen schnelle Demontage der beschichtete Gruben an der Plasmamembran induziert und Darstellung neuer blockiert. Zusatz von exogenen PIP(2) kehrt diesen Effekt. Beschichtete Gruben und Bl&auml;schen wieder synchron erscheinen nach der Entfernung von 1-Butanol; Wir haben diese Synchronit&auml;t verwendet, um die Rolle der Aktin in beschichteten Vesikel-Assembly zu bewerten. Anhaltende Hemmung der Aktin-Polymerisation von Latrunculin A oder Cytochalasin D reduziert um ca. 50 % die H&auml;ufigkeit der beschichteten Grube Bildung ohne Reifung in beschichteten Bl&auml;schen. Wie im Steuerelement Zellen f&uuml;hrte Entfernung von 1-Butanol in die weitere Pr&auml;senz der ein Aktin-Depolymerizer mit synchroner Darstellung der neuen Gruben, die normalerweise f&auml;llig. Also Umbau des Aktin-Zytoskeletts sind nicht f&uuml;r Clathrin-coated Vesikel Versammlung sondern kann indirekt beeinflussen die Erforschung der Clathrin-beschichtete Gruben.

Rolle der Lipide und Actin in der Bildung von Clathrin-beschichtete Gruben.

Assemblea delle cave rivestite da clatrina e loro maturazione in vescicole rivestite richiede interazioni coordinate tra specifici lipidi e proteine strutturali e normative diversi. In presenza di alcoli primari, fosfolipasi D genera phosphatidylalcohols anzich&eacute; PA, riducendo la stimolazione del fosfatidil inositolo 5-chinasi (PI5K) e quindi diminuendo la formazione di phosphoinositide-4,5-biphosphate (PIP(2)). Usando live cell imaging, abbiamo dimostrato che trattamento acuto delle cellule con 1-butanolo o di altri alcoli primari piccoli induce lo smontaggio rapido dei pozzi rivestiti in membrana del plasma e blocca la comparsa di nuove. Aggiunta di PIP(2) esogeno inverte questo effetto. Le vescicole e pozzi rivestiti riappariranno in modo sincrono dopo l&#39;espianto di 1-butanolo; abbiamo usato questa sincronia di valutare il ruolo dell&#39;actina in Assemblea vescicola rivestita. Prolungata inibizione della polimerizzazione dell&#39;actina di latrunculin A o citocalasina D ridotto di circa il 50% la frequenza di formazione fossa rivestita senza interessare la maturazione in vescicole rivestite. Come nelle cellule di controllo, rimozione di 1-butanolo nella presenza costante di un depolymerizer di actina ha portato alla comparsa sincrona di nuovi pozzi, che ha maturato normalmente. Cos&igrave;, rimodellamento del citoscheletro di actina non &egrave; essenziale per l&#39;assemblaggio della vescicola rivestita da clatrina ma indirettamente possa influenzare la nucleazione dei pozzi rivestite da Clatrina.

Ruolo dei lipidi e dell&#39;actina nella formazione di cavit&agrave; rivestite da Clatrina.

&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x30EA;&#x30F3;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x3068;&#x305D;&#x308C;&#x3089;&#x306E;&#x6210;&#x719F;&#x88AB;&#x8986;&#x5C0F;&#x80DE;&#x306B;&#x5354;&#x8ABF;&#x3068;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x7279;&#x5B9A;&#x8102;&#x8CEA;&#x69CB;&#x9020;&#x3068;&#x898F;&#x5236;&#x306E;&#x3044;&#x304F;&#x3064;&#x304B;&#x306E;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x304C;&#x5FC5;&#x8981;&#x3067;&#x3059;&#x3002;&#x7B2C;&#x4E00;&#x7D1A;&#x30A2;&#x30EB;&#x30B3;&#x30FC;&#x30EB;&#x5B58;&#x5728;&#x4E0B;&#x306B;&#x304A;&#x3051;&#x308B;&#x30DB;&#x30B9;&#x30DB;&#x30EA;&#x30D1;&#x30FC;&#x30BC; D phosphatidylalcohols PA &#x306E;&#x30DB;&#x30B9;&#x30D5;&#x30A1;&#x30C1;&#x30B8;&#x30EB;&#x30B3;&#x30EA;&#x30F3; &#x30A4;&#x30CE;&#x30B7;&#x30C8;&#x30FC;&#x30EB; 5 &#x30AD;&#x30CA;&#x30FC;&#x30BC; (PI5K) &#x306E;&#x523A;&#x6FC0;&#x3092;&#x524A;&#x6E1B;&#x3057;&#x3001;&#x305D;&#x308C;&#x3086;&#x3048;&#x30DB;&#x30B9;&#x30DB;&#x30A4;&#x30CE;&#x30B7;&#x30C1;&#x30C9;-4, 5-biphosphate &#xFF08;PIP(2)). &#x306E;&#x5F62;&#x6210;&#x3092;&#x6E1B;&#x5C11;&#x3055;&#x305B;&#x308B;&#x306E;&#x3067;&#x306F;&#x306A;&#x304F;&#x751F;&#x6210;&#x3057;&#x307E;&#x3059;&#x3002;&#x4F4F;&#x30BB;&#x30EB;&#x30A4;&#x30E1;&#x30FC;&#x30B8;&#x6295;&#x5C04;&#x3092;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3001;&#x6211;&#x3005; &#x306F; 1-&#x30D6;&#x30BF;&#x30CE;&#x30FC;&#x30EB;&#x307E;&#x305F;&#x306F;&#x4ED6;&#x306E;&#x5C0F;&#x3055;&#x306A;&#x7B2C;&#x4E00;&#x7D1A;&#x30A2;&#x30EB;&#x30B3;&#x30FC;&#x30EB;&#x3068;&#x7D30;&#x80DE;&#x306E;&#x6025;&#x6027;&#x671F;&#x6CBB;&#x7642;&#x306F;&#x539F;&#x5F62;&#x8CEA;&#x819C;&#x306E;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x306E;&#x6025;&#x901F;&#x306A;&#x5206;&#x89E3;&#x3092;&#x8A98;&#x5C0E;&#x3057;&#x3066;&#x65B0;&#x3057;&#x3044;&#x3082;&#x306E;&#x306E;&#x5916;&#x89B3;&#x3092;&#x30D6;&#x30ED;&#x30C3;&#x30AF;&#x3092;&#x793A;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;&#x5916;&#x56E0;&#x6027; PIP(2) &#x306E;&#x6DFB;&#x52A0;&#x306F;&#x3001;&#x3053;&#x306E;&#x52B9;&#x679C;&#x3092;&#x53CD;&#x8EE2;&#x3055;&#x305B;&#x307E;&#x3059;&#x3002;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x3068;&#x5C0F;&#x80DE; 1-&#x30D6;&#x30BF;&#x30CE;&#x30FC;&#x30EB;&#x306E;&#x9664;&#x53BB;&#x6642;&#x306B;&#x518D;&#x540C;&#x671F;&#x7684;&#x306B;;&#x6211;&#x3005; &#x306F;&#x3053;&#x306E;&#x540C;&#x8ABF;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x88AB;&#x8986;&#x5C0F;&#x80DE;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306E;&#x5F79;&#x5272;&#x3092;&#x8A55;&#x4FA1;&#x3059;&#x308B;&#x305F;&#x3081;&#x306B;&#x4F7F;&#x7528;&#x3057;&#x3066;&#x3044;&#x307E;&#x3059;&#x3002;Latrunculin A &#x307E;&#x305F;&#x306F;&#x30B5;&#x30A4;&#x30C8;&#x30AB;&#x30E9;&#x30B7;&#x30F3; D &#x306B;&#x3088;&#x308B;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x91CD;&#x5408;&#x306E;&#x9577;&#x671F;&#x6291;&#x5236;&#x7D04; 50 &#xFF05; &#x306F;&#x88AB;&#x8986;&#x5C0F;&#x80DE;&#x306B;&#x6210;&#x719F;&#x306B;&#x5F71;&#x97FF;&#x3092;&#x4E0E;&#x3048;&#x305A;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x5F62;&#x6210;&#x306E;&#x983B;&#x5EA6;&#x3092;&#x6E1B;&#x3089;&#x3057;&#x307E;&#x3059;&#x3002;&#x30B3;&#x30F3;&#x30C8;&#x30ED;&#x30FC;&#x30EB;&#x306E;&#x30BB;&#x30EB;&#x3068;&#x540C;&#x69D8;&#x306B; 1-&#x30D6;&#x30BF;&#x30CE;&#x30FC;&#x30EB; &#x30A2;&#x30AF;&#x30C1;&#x30F3; depolymerizer &#x306E;&#x7D99;&#x7D9A;&#x7684;&#x306A;&#x5B58;&#x5728;&#x3067;&#x9664;&#x53BB;&#x901A;&#x5E38;&#x6210;&#x719F;&#x65B0;&#x3057;&#x3044;&#x30D4;&#x30C3;&#x30C8;&#x306E;&#x540C;&#x671F;&#x306E;&#x5916;&#x89B3;&#x306B;&#x306A;&#x3063;&#x305F;&#x3002;&#x3057;&#x305F;&#x304C;&#x3063;&#x3066;&#x3001;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x7D30;&#x80DE;&#x9AA8;&#x683C;&#x306E;&#x30EA;&#x30E2;&#x30C7;&#x30EA;&#x30F3;&#x30B0; &#x30AF;&#x30E9;&#x30B9;&#x30EA;&#x30F3;&#x88AB;&#x8986;&#x5C0F;&#x80DE;&#x306E;&#x30A2;&#x30BB;&#x30F3;&#x30D6;&#x30EA;&#x306B;&#x5FC5;&#x9808;&#x3067;&#x306F;&#x3042;&#x308A;&#x307E;&#x305B;&#x3093;&#x304C;&#x3001;&#x76F4;&#x63A5;&#x6838;&#x5F62;&#x6210;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x30EA;&#x30F3;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x306B;&#x5F71;&#x97FF;&#x53EF;&#x80FD;&#x6027;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3002;

&#x8102;&#x8CEA;&#x3068;&#x30A2;&#x30AF;&#x30C1;&#x30F3;&#x306E;&#x30AF;&#x30E9;&#x30B9;&#x30EA;&#x30F3;&#x88AB;&#x8986;&#x30D4;&#x30C3;&#x30C8;&#x5F62;&#x6210;&#x3067;&#x306E;&#x5F79;&#x5272;&#x3002;

&#xAD6C; &#xB369;&#xC774; clathrin &#xCF54;&#xD305; &#xBC0F; &#xCF54;&#xD305;&#xB41C; &#xC18C;&#xD3EC;&#xC5D0; &#xADF8;&#xB4E4;&#xC758; &#xC131;&#xC219;&#xC758; &#xD2B9;&#xC815; &#xC9C0;&#xC9C8;&#xACFC; &#xC5EC;&#xB7EC; &#xAC00;&#xC9C0; &#xAD6C;&#xC870;&#xC640; &#xADDC;&#xC81C; &#xB2E8;&#xBC31;&#xC9C8; &#xAC04;&#xC758; &#xD611;&#xC870; &#xC0C1;&#xD638; &#xC791;&#xC6A9; &#xD574;&#xC57C;&#xD569;&#xB2C8;&#xB2E4;. &#xAE30;&#xBCF8; &#xC54C;&#xCF5C;&#xC758; &#xC874;&#xC7AC; phospholipase D PA, phosphatidyl inositol 5 &#xD0A4; (PI5K)&#xC758; &#xC790;&#xADF9;&#xC744; &#xAC10;&#xC18C; &#xD558; &#xACE0; &#xB530;&#xB77C;&#xC11C; phosphoinositide-4, 5-biphosphate (PIP(2))&#xC758; &#xD615;&#xC131;&#xC744; &#xAC10;&#xC18C; &#xD558;&#xB294; &#xB300;&#xC2E0; phosphatidylalcohols&#xB97C; &#xC0DD;&#xC131;. &#xB77C;&#xC774;&#xBE0C; &#xC140; &#xC774;&#xBBF8;&#xC9D5; &#xC0AC;&#xC6A9; &#xD558; &#xC5EC;, &#xC6B0;&#xB9AC;&#xAC00; &#xB098;&#xD0C0;&#xB0AC;&#xC2B5;&#xB2C8;&#xB2E4; &#xC140; 1 butanol &#xB610;&#xB294; &#xB2E4;&#xB978; &#xC791;&#xC740; 1 &#xCC28; &#xC54C;&#xCF54;&#xC62C;&#xC758; &#xAE09;&#xC131; &#xCE58;&#xB8CC; &#xC6D0;&#xD615;&#xC9C8; &#xB9C9;&#xC5D0; &#xCF54;&#xD305; &#xB41C; &#xAD6C; &#xB369;&#xC774;&#xC758; &#xAE09;&#xC18D; &#xD55C; &#xBD84;&#xD574;&#xB97C; &#xC720;&#xB3C4; &#xD558; &#xACE0; &#xC0C8;&#xB85C;&#xC6B4; &#xAC83; &#xB4E4;&#xC758; &#xBAA8;&#xC2B5;&#xC744; &#xCC28;&#xB2E8;. Exogenous PIP(2)&#xC758; &#xCD94;&#xAC00;&#xC774; &#xD6A8;&#xACFC; &#xBC18;&#xB300;&#xB85C; &#xBC14;&#xAFC9;&#xB2C8;&#xB2E4;. &#xCF54;&#xD305;&#xB41C; &#xAD6C; &#xB369;&#xC774; &#xC18C;&#xD3EC; 1-butanol;&#xC758; &#xC81C;&#xAC70;&#xC5D0; &#xB530;&#xB77C; &#xB3D9;&#xAE30;&#xC801;&#xC73C;&#xB85C; &#xB2E4;&#xC2DC; &#xC6B0;&#xB9AC;&#xAC00;&#xC774; synchrony &#xCF54;&#xD305;&#xB41C; &#xAE30; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0;&#xC11C; &#xB9D0;&#xB77C;&#xC758; &#xC5ED;&#xD560;&#xC744; &#xD3C9;&#xAC00; &#xD558;&#xAE30; &#xC704;&#xD574; &#xC0AC;&#xC6A9;. &#xC7A5;&#xAE30;&#xAC04;&#xB41C; &#xC5B5;&#xC81C; &#xB9D0;&#xB77C; &#xD569; latrunculin A &#xB610;&#xB294; D cytochalasin&#xC758; &#xC57D; 50% &#xCF54;&#xD305;&#xB41C; vesicles&#xB85C; &#xC131;&#xC219;&#xC5D0; &#xC601;&#xD5A5;&#xC744; &#xC8FC;&#xC9C0; &#xC54A;&#xACE0; &#xCF54;&#xD305;&#xB41C; &#xAD6C; &#xB369;&#xC774; &#xD615;&#xC131;&#xC758; &#xBE48;&#xB3C4;&#xAC00; &#xAC10;&#xC18C;. &#xC81C;&#xC5B4; &#xC140;&#xC5D0;&#xC11C; &#xC81C;&#xAC70; 1-butanol &#xB9D0;&#xB77C; depolymerizer&#xC758; &#xC9C0;&#xC18D;&#xC801;&#xC778;&#xB41C; &#xC874;&#xC7AC;&#xC5D0;&#xC758; &#xC815;&#xC0C1;&#xC801;&#xC73C;&#xB85C; &#xC131;&#xC219; &#xD558;&#xB294; &#xC0C8;&#xB85C;&#xC6B4; &#xAD6C; &#xB369;&#xC774;&#xC758; &#xB3D9;&#xAE30; &#xBAA8;&#xC591;&#xC744; &#xC774;&#xB04C;&#xC5B4; &#xB0C8;&#xB2E4;. &#xB530;&#xB77C;&#xC11C;, &#xB9D0;&#xB77C; cytoskeleton&#xC758; &#xAC1C;&#xC7A5; clathrin-&#xC785;&#xD788;&#xB294; &#xC18C;&#xD3EC; &#xC5B4;&#xC148;&#xBE14;&#xB9AC;&#xC5D0; &#xB300; &#xD55C; &#xC911;&#xC694; &#xD558;&#xC9C0; &#xC54A;&#xC2B5;&#xB2C8;&#xB2E4; &#xD558;&#xC9C0;&#xB9CC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xC9C1;&#xC811; clathrin &#xCF54;&#xD305; &#xAD6C; &#xB369;&#xC774;&#xC758; nucleation &#xC601;&#xD5A5;.

&#xC9C0;&#xC9C8;&#xACFC; &#xB9D0;&#xB77C; &#xAD6C; &#xB369;&#xC774; clathrin &#xCF54;&#xD305; &#xD615;&#xC131;&#xC5D0;&#xC758; &#xC5ED;&#xD560;.

Asamblea de hoyos recubiertos de clatrina y su maduraci&oacute;n en ves&iacute;culas recubiertas requiere interacciones coordinadas entre los l&iacute;pidos espec&iacute;ficos y varias prote&iacute;nas estructurales y reguladoras. En presencia de alcoholes primarios, fosfolipasa D genera phosphatidylalcohols en lugar de PA, reducci&oacute;n de la estimulaci&oacute;n de fosfatidil inositol 5-quinasa (PI5K) y por lo tanto disminuir la formaci&oacute;n de Fosfatidilinositol-4, 5-bifosfato (PIP(2)). Usando la proyecci&oacute;n de imagen de c&eacute;lulas vivas, hemos demostrado que el tratamiento agudo de c&eacute;lulas con alcoholes primarios peque&ntilde;os 1-butanol u otros induce desmontaje r&aacute;pido de Tunica en la membrana plasm&aacute;tica y bloquea la aparici&oacute;n de otras nuevas. Adici&oacute;n de PIP(2) ex&oacute;geno revierte este efecto. Ves&iacute;culas y Tunica aparecer sincr&oacute;nicamente al retirar de 1-butanol; hemos utilizado esta sincron&iacute;a para evaluar el papel de la actina en Asamblea de ves&iacute;culas revestidas. Inhibici&oacute;n prolongada de la polimerizaci&oacute;n de la actina por LATRUNCULINA A o Citocalasina D hab&iacute;a reducido en aproximadamente un 50% la frecuencia de formaci&oacute;n de Hoyo revestido sin afectar a la maduraci&oacute;n en ves&iacute;culas revestidas. Como en las c&eacute;lulas control, eliminaci&oacute;n de 1-butanol en la permanencia de un depolymerizer de actina condujo al aspecto sincr&oacute;nico de nuevos pozos, que madur&oacute; normalmente. As&iacute;, remodelaci&oacute;n del citoesqueleto de actina no es esencial para el montaje de ves&iacute;culas recubiertas de clatrina pero puede afectar indirectamente la nucleaci&oacute;n de hoyos recubiertos de clatrina.

Papel de los l&iacute;pidos y la actina en la formaci&oacute;n de hoyos recubiertos de clatrina.

Recent evidence suggests that the EGF receptor oligomerizes or clusters in cells even in the absence of agonist ligand. To assess the status of EGF receptors in live cells, an EGF receptor fused to eGFP was stably expressed in CHO cells and studied using fluorescence correlation spectroscopy and fluorescent brightness analysis. By modifying FIDA for use in a two-dimensional system with quantal brightnesses, a method was developed to quantify the degree of clustering of the receptors on the cell surface. The analysis demonstrates that under physiological conditions, the EGF receptor exists in a complex equilibrium involving single molecules and clusters of two or more receptors. Acute depletion of cellular cholesterol enhanced EGF receptor clustering whereas cholesterol loading decreased receptor clustering, indicating that receptor aggregation is sensitive to the lipid composition of the membrane.

Oligomerization of the EGF receptor investigated by live cell fluorescence intensity distribution analysis.

We present a method to resolve components within a diffraction-limited object by tracking simultaneously the average axial positions of two different sets of fluorescent molecules within it. The axial positions are then subtracted from each other to determine the separation of the two sets of fluorophores. This method follows the dynamic changes in the separation of the two sets of fluorophores with freely rotating dipoles using sequential acquisitions with total internal reflection and wide-field illumination, and it can be used to measure the formation of small structures on living cells. We have verified that we can achieve a resolution of 10 nm, and we have used the method to follow the location of clathrin and its adaptor AP-2 as they are recruited to a diffraction-limited coated pit during its assembly at the plasma membrane. We find a gradually increasing axial separation between the centroids of clathrin and AP-2 distribution, up to a final value of 30 nm just before coated-pit pinching and formation of the coated vesicle.

Differential evanescence nanometry: live-cell fluorescence measurements with 10-nm axial resolution on the plasma membrane.

Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.

Vesicular stomatitis virus enters cells through vesicles incompletely coated with clathrin that depend upon actin for internalization.

Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as "coated pits" and "coated plaques." Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane--classical coated pits and coated plaques--and that these two assemblies interact quite differently with other intracellular structures.

Distinct dynamics of endocytic clathrin-coated pits and coated plaques.

The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.

Roles of AP-2 in clathrin-mediated endocytosis.

&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#x50AC;&#x5316;&#x7684;&#x7EC6;&#x80DE;&#x5916;&#x57FA;&#x8D28;&#x91CD;&#x6784;&#x662F;&#x5728;&#x5F00;&#x53D1;&#x8FC7;&#x7A0B;&#x4E2D;&#x5F62;&#x6001;&#x53D1;&#x751F;&#x73B0;&#x8C61;&#x7684;&#x6838;&#x5FC3;&#x548C;&#x4F24;&#x53E3;&#x6108;&#x5408;&#xFF0C;&#x4EE5;&#x53CA;&#x5728;&#x8BB8;&#x591A;&#x75C5;&#x7406;&#x6761;&#x4EF6;&#x4E0B;&#x7EA4;&#x7EF4;&#x5316;&#x548C;&#x764C;&#x75C7;&#x7B49;&#x3002;&#x6211;&#x4EEC;&#x4EE5;&#x524D;&#x5DF2;&#x7ECF;&#x663E;&#x793A;&#x5206;&#x6CCC;&#x7684;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2 &#x662F;&#x62F4;&#x5728;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#xFF0C;&#x5E76;&#x901A;&#x8FC7; MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;/&#x86CB;&#x767D;&#x9176;-2 &#x7684;&#x4F9D;&#x8D56;&#x673A;&#x5236;&#x6FC0;&#x6D3B;&#x3002;&#x7531;&#x6B64;&#x4EA7;&#x751F;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x80F6;&#x539F;&#x590D;&#x6742; (MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#xFF09; 2/&#x86CB;&#x767D;&#x9176;-2/&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2 &#x53EF;&#x4EE5;&#x542F;&#x52A8; (MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;) &#x548C;&#x5B8C;&#x6210; (&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2) &#x9000;&#x5316;&#x7684;&#x57FA;&#x7840;&#x7684;&#x80F6;&#x539F;&#x7EA4;&#x7EF4;&#x3002;&#x4E0B;&#x9762;&#x7684;&#x95EE;&#x9898;&#x4ECD;&#x7136;&#x662F;&#xFF1A; &#x76F8;&#x5BF9;&#x4E0D;&#x4FBF;&#x3001; &#x7EC6;&#x80DE;&#x8868;&#x9762;&#x9176;&#x590D;&#x5408;&#x7269;&#x548C;&#x80F6;&#x539F;&#x7EA4;&#x7EF4;&#x7684;&#x4E24;&#x79CD;&#x7ED3;&#x6784;&#x7684;&#x5E95;&#x7269;&#x8BC6;&#x522B;&#x6D89;&#x53CA;&#x7684;&#x673A;&#x5236;&#x662F;&#x4EC0;&#x4E48;&#x5D4C;&#x5165;&#x5728; ECM &#x5417;&#xFF1F;&#x5728;&#x8FD9;&#x91CC;&#x6211;&#x4EEC;&#x5C55;&#x793A;&#x7684;&#x590D;&#x6742;&#x7684;&#x6240;&#x6709;&#x7EC4;&#x4EF6;&#x90FD;&#x90FD;&#x80FD;&#x591F;&#x845B;&#x8FD0;&#x52A8;&#x7684;&#x8868;&#x9762;&#x4E0A;&#x7684;&#x80F6;&#x539F;&#x7EA4;&#x7EF4;&#x3002;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-MT1 &#x8FD0;&#x52A8;&#x7684;&#x673A;&#x5236;&#x662F;&#x6709;&#x504F;&#x89C1;&#x7684;&#x4F20;&#x64AD;&#x4E0E;&#x504F;&#x7F6E;&#x7EC4;&#x4EF6;&#x4F9D;&#x8D56;&#x4E8E;&#x86CB;&#x767D;&#x8D28;&#x5206;&#x89E3;&#x5176;&#x886C;&#x5E95;&#xFF0C;&#x4E09;&#x78F7;&#x9178;&#x817A;&#x82F7; (ATP) &#x4E0D;&#x6C34;&#x89E3;&#x3002;&#x5B83;&#x662F;&#x7C7B;&#x4F3C;&#x4E8E;&#x7684;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-1 &#x5E03;&#x6717;&#x9010;&#x6B65;&#x6211;&#x4EEC;&#x524D;&#x9762;&#x6240;&#x8FF0;&#x3002;&#x6B64;&#x5916;&#xFF0C;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2 &#x548C;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-9 &#x4EE5;&#x53CA;&#x86CB;&#x767D;&#x9176;-1 &#x548C;-2 &#x53CA;&#x5176;&#x5404;&#x81EA;&#x914D;&#x5408;&#x800C;&#x80FD;&#x591F;&#x5E03;&#x6717;&#x6269;&#x6563;&#x8868;&#x9762;&#x65E0;&#x660E;&#x663E;&#x5206;&#x79BB;&#x672C;&#x673A;&#x7684;&#x80F6;&#x539F;&#x539F;&#x7EA4;&#x7EF4;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-9 &#x7684;&#x4E8C;&#x805A;&#x53CD;&#x5E94;&#x5448;&#x73B0;&#x8FD9;&#x79CD;&#x9176;&#x4E0D;&#x52A8;&#x3002;&#x6700;&#x6709;&#x542F;&#x53D1;&#x6027;&#x7684;&#x662F;&#x706D;&#x6D3B; MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#x7684;&#x9176;&#x7684;&#x6D3B;&#x6027;&#x6709;&#x5173;&#x4E8E;&#x5355;&#x5143;&#x683C;&#x90E8;&#x961F;&#x5236;&#x5B9A;&#x7684;&#x5C0F;&#x578B;&#x5316; 3D &#x7EC4;&#x7EC7;&#x6784;&#x9020;&#x63A2;&#x6D4B;&#x7684;&#x8D1F;&#x9762;&#x5F71;&#x54CD;&#xFF0C;&#x8FD9;&#x4E00;&#x53D1;&#x73B0;&#x3002;&#x6211;&#x4EEC;&#x5EFA;&#x8BAE;&#x80F6;&#x539F;&#x86CB;&#x767D;&#x590D;&#x6742; (MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#xFF09; 2/&#x86CB;&#x767D;&#x9176;-2/&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2 &#x8868;&#x793A;&#x79FB;&#x52A8;&#x5355;&#x5143;&#x683C;&#x8868;&#x9762;&#x80F6;&#x539F;&#x57FA;&#x8D28;&#x754C;&#x9762;&#x3002;MT1-&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#x4F5C;&#x4E3A;&#x62F4;&#x5728;&#x590D;&#x6742;&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;-2 &#x4E0E;&#x7EC6;&#x80DE;&#x8868;&#x9762;&#x7684;&#x5206;&#x5B50;&#x68D8;&#x8F6E;&#x5BF9;&#x751F;&#x7269;&#x7684;&#x5F71;&#x54CD;&#x5EFA;&#x8BAE;&#x7684;&#x7A7A;&#x95F4;&#x53D7;&#x89C4;&#x7BA1;&#x56F4;&#x7EC6;&#x80DE;&#x86CB;&#x767D;&#x964D;&#x89E3;&#x7EC6;&#x80DE;&#x57FA;&#x8D28;&#x4EA4;&#x4E92;&#x4F5C;&#x7528;&#x7684;&#x65B0;&#x673A;&#x5236;&#x3002;

&#x57FA;&#x8D28;&#x91D1;&#x5C5E;&#x86CB;&#x767D;&#x9176;&#x7684;&#x80F6;&#x539F;&#x539F;&#x7EA4;&#x7EF4;&#x8868;&#x9762;&#x6269;&#x6563;&#xFF1A; &#x79FB;&#x52A8;&#x5355;&#x5143;&#x683C;&#x8868;&#x9762;&#x80F6;&#x539F;&#x57FA;&#x8D28;&#x63A5;&#x53E3;&#x3002;

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)(2)/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)(2)/TIMP-2/MMP-2 represents a Mobile Cell Surface-Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

Remodelage de la matrice extracellulaire catalys&eacute;e par MMP est au c&oelig;ur des ph&eacute;nom&egrave;nes morphog&eacute;n&eacute;tiques au cours du d&eacute;veloppement et enroul&eacute; de gu&eacute;rison ainsi que de nombreuses conditions pathologiques telles que la fibrose et le cancer. Nous avons d&eacute;montr&eacute; pr&eacute;c&eacute;demment que s&eacute;cr&eacute;t&eacute;es MMP-2 est attach&eacute; &agrave; la surface des cellules et activ&eacute; par le m&eacute;canisme de la MT1-MMP/TIMP-2-d&eacute;pendante. La r&eacute;sultante collag&eacute;nolytique surface cellulaire complexe (MT1-MMP) 2/TIMP-2/MMP-2 peut initier (MT1-MMP) et achever la d&eacute;gradation d&#39;une fibrille de collag&egrave;ne sous-jacent (MMP-2). Reste la question suivante : quel est le m&eacute;canisme de la reconnaissance du substrat impliquant les deux structures de mobilit&eacute; relativement restreinte, le complexe enzymatique surface cellulaire et une fibrille collag&egrave;ne incorpor&eacute; dans l&#39;ECM ? Nous d&eacute;montrons que toutes les composantes du complexe sont capables de mouvements processive sur une surface de la fibrille de collag&egrave;ne. Le m&eacute;canisme du mouvement de la MT1-MMP est une diffusion biais&eacute;e avec la composante de polarisation d&eacute;pend de la prot&eacute;olyse de son substrat, l&#39;hydrolyse de l&#39;ad&eacute;nosine triphosphate (ATP) pas. Il est similaire &agrave; que de la MMP-1 brownienne ratchet nous d&eacute;crit pr&eacute;c&eacute;demment. En outre, MMP-2 et MMP-9, ainsi que leurs complexes respectifs avec TIMP-1 et -2 sont capables de diffusion brownienne sur la surface des fibrilles de collag&egrave;ne natif sans dissociation perceptible alors que la dim&eacute;risation de MMP-9 rend l&#39;enzyme immobile. Plus instructive est la constatation que l&#39;inactivation de l&#39;activit&eacute; enzymatique de la MT1-MMP a un effet n&eacute;gatif d&eacute;tectable sur la force de cellules d&eacute;velopp&eacute;e dans les constructions de tissu 3D miniaturis&eacute;s. Nous proposons que le collag&eacute;nolytique complexes (MT1-MMP) 2/TIMP-2/MMP-2 repr&eacute;sente une Interface de substrat de Surface-collag&egrave;ne Mobile cellulaire. Les cons&eacute;quences biologiques de la MT1-MMP, agissant comme un cliquet mol&eacute;culaire attach&eacute; &agrave; la surface de la cellule en complexe avec MMP-2 propose un nouveau m&eacute;canisme pour le r&ocirc;le de la prot&eacute;olyse de peri-cellulaires dans l&#39;espace r&eacute;glement&eacute; dans les interactions cellule-matrice.

Diffusion des MPM sur la surface des fibrilles de collag&egrave;ne : l&#39;interface de substrat de surface-collag&egrave;ne mobile cellulaire.

Umbau der extrazellul&auml;ren Matrix, katalysiert durch MMPs ist morphogenetische Ph&auml;nomene w&auml;hrend der Entwicklung und Wunde heilenden sowie in zahlreichen pathologische Bedingungen wie Fibrose und Krebs. Wir haben bereits bewiesen, dass sezernierte MMP-2 auf der Zelloberfl&auml;che angebunden und von MT1-MMP/TIMP-2-abh&auml;ngige Mechanismus aktiviert ist. Die resultierende Zelloberfl&auml;che Collagenolytic Komplex (MT1-MMP) 2/TIMP-2/MMP-2 kann (MT1-MMP) zu initiieren und (MMP-2) Verschlechterung der eine zugrunde liegende Kollagen-Fibrille. Die folgende Frage blieb: Was ist der Mechanismus der Substrat-Anerkennung mit beiden Strukturen relativ eingeschr&auml;nkte Beweglichkeit, der Zelle Oberfl&auml;che enzymatischen komplex und eine Kollagen-Fibrille eingebettet in das ECM? Hier zeigen wir, dass alle Komponenten des Komplexes prozessualen Bewegung auf einer Fl&auml;che von Kollagen-Fibrille f&auml;hig sind. Der Mechanismus der MT1-MMP-Bewegung ist eine voreingenommene Diffusion mit der Belichtungszeit-Komponente der Proteolyse abh&auml;ngig sein Substrat, nicht Adenosintriphosphat (ATP) Hydrolyse. Es ist &auml;hnlich, dass der MMP-1 Brownsche Ratsche wir wie oben beschrieben. Dar&uuml;ber hinaus k&ouml;nnen sowohl MMP-2 und MMP-9 sowie ihre jeweiligen komplexe mit TIMP-1 und-2 Brownschen Verbreitung auf der Oberfl&auml;che der native Kollagen-Fibrillen ohne merkliche Dissoziation w&auml;hrend die Dimerisierung von MMP-9 das Enzym bewegungsunf&auml;hig macht. Sehr aufschlussreich ist die Feststellung, die die Inaktivierung der enzymatischen Aktivit&auml;t der MT1-MMP nachweisbar negativ auf die Zelle Kraft in miniaturisierten 3D Gewebe Konstrukte entwickelt hat. Wir schlagen die Collagenolytic Komplex (MT1-MMP) 2/TIMP-2/MMP-2 eine Mobile Zelle Oberfl&auml;che-Collagen-Substrat-Schnittstelle darstellt. Die biologischen Auswirkungen der MT1-MMP als eine molekulare Ratsche angebunden an der Zelloberfl&auml;che im Komplex mit MMP-2 schlagen einen neuen Mechanismus f&uuml;r die Rolle der r&auml;umlich geregelten Peri / Mobilfunk Proteolyse in Zelle-Matrix-Interaktionen.

Verbreitung der MMPs auf der Oberfl&auml;che von Kollagen-Fibrillen: die mobile Zelle Oberfl&auml;che-Collagen-Substrat-Schnittstelle.

Rimodellamento della matrice extracellulare catalizzata da MMP &egrave; centrale a fenomeni morfogenetici durante lo sviluppo e la ferita guarigione cos&igrave; come in numerose condizioni patologiche quali fibrosi e cancro. Abbiamo precedentemente dimostrato che secrete MMP-2 &egrave; legato alla superficie delle cellule e attivato dal meccanismo MT1-MMP/TIMP-2-dipendente. La superficie della cellula risultante collagenolytic di complessi (MT1-MMP) (2) / TIMP-2/MMP-2 possono avviare (MT1-MMP) e completare la degradazione (MMP-2) di un sottostante fibrillare del collagene. Rimase la seguente domanda: Qual &egrave; il meccanismo di riconoscimento del substrato che coinvolgono le due strutture di mobilit&agrave; relativamente ristretto, il complesso enzimatico superficie cellulare e una fibrillare collagene incorporato in ECM? Qui noi dimostrare che tutti i componenti del complesso sono in grado di processive movimento su una superficie di fibrillare del collagene. Il meccanismo di movimento MT1-MMP &egrave; una diffusione distorta con il componente bias dipende la proteolisi del suo substrato, non all&#39;idrolisi dell&#39;adenosina trifosfato (ATP). &Egrave; simile alla che di browniano di MMP-1 cricchetto abbiamo descritto in precedenza. Inoltre, sia MMP-2 e MMP-9, nonch&eacute; i rispettivi complessi con TIMP-1 e -2 sono in grado di diffusione browniana sulla superficie delle fibrille di collagene nativo senza evidente dissociazione mentre la dimerizzazione della MMP-9 rende l&#39;enzima immobile. Pi&ugrave; istruttiva &egrave; la constatazione che l&#39;inattivazione dell&#39;attivit&agrave; enzimatica della MT1-MMP ha un effetto negativo rilevabile sulla cella forza sviluppata in tessuto 3D miniaturizzato costrutti. Proponiamo che il complesso di collagenolytic (MT1-MMP) (2) / TIMP-2/MMP-2 rappresenta un&#39;interfaccia di substrato Mobile Cell superficie-collagene. Le implicazioni biologiche di MT1-MMP che agisce come una leva a cricco molecolare legato alla superficie delle cellule in complesso con MMP-2 suggeriscono un nuovo meccanismo per il ruolo della proteolisi peri-cellulare spazialmente regolamentata nelle interazioni cellula-matrice.

Diffusione di MMP sulla superficie delle fibrille di collagene: l&#39;interfaccia di superficie-collagene substrato mobile cell.

Mmp &#x306B;&#x3088;&#x308B;&#x89E6;&#x5A92;&#x306B;&#x3088;&#x308B;&#x7D30;&#x80DE;&#x5916;&#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x6539;&#x9020;&#x958B;&#x767A;&#x6642;&#x306B;&#x5F62;&#x614B;&#x5F62;&#x6210;&#x73FE;&#x8C61;&#x306B;&#x4E2D;&#x592E;&#x3067;&#x3059;&#x3001;&#x5275;&#x50B7;&#x6CBB;&#x7652;&#x3060;&#x3051;&#x3067;&#x306A;&#x304F;&#x3001;&#x7DDA;&#x7DAD;&#x75C7;&#x3084;&#x304C;&#x3093;&#x306A;&#x3069;&#x306E;&#x591A;&#x6570;&#x306E;&#x75C5;&#x7406;&#x5B66;&#x7684;&#x6761;&#x4EF6;&#x3002;&#x6211;&#x3005; &#x306F;&#x4EE5;&#x524D;&#x5206;&#x6CCC; MMP 2 &#x304C;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x4FC2;&#x7559;&#x3055;&#x308C; MT1 MMP/&#x30C1;&#x30F3;&#x30D7; 2 &#x4F9D;&#x5B58;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306B;&#x3088;&#x3063;&#x3066;&#x6D3B;&#x6027;&#x5316;&#x3092;&#x5B9F;&#x8A3C;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x7D50;&#x679C;&#x306E;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x30B3;&#x30E9;&#x30B2;&#x30CA;&#x30FC;&#x30BC;&#x3092;&#x8907;&#x96D1;&#x306A; &#xFF08;MT1 MMP&#xFF09; (2)/&#x30C1;&#x30F3;&#x30D7; 2/MMP 2 &#x3059;&#x308B;&#x3053;&#x3068;&#x304C;&#x3067;&#x304D;&#x307E;&#x3059; &#xFF08;MT1 MMP&#xFF09; &#x3092;&#x958B;&#x59CB;&#x3057;&#x3001;&#xFF08;MMP-2) &#x5206;&#x89E3;&#x306F;&#x57FA;&#x306B;&#x306A;&#x308B;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x7D30;&#x7DDA;&#x7DAD;&#x306E;&#x5B8C;&#x4E86;&#x3057;&#x307E;&#x3059;&#x3002;&#x6B21;&#x306E;&#x8CEA;&#x554F;&#x306B;&#x6B8B;&#x3063;&#x305F;: &#x542B;&#x3080;&#x57FA;&#x8CEA;&#x8A8D;&#x8B58;&#x306E;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x306F;&#x4F55;&#x3067;&#x3059;&#x6BD4;&#x8F03;&#x7684;&#x5236;&#x9650;&#x3055;&#x308C;&#x305F;&#x30E2;&#x30D3;&#x30EA;&#x30C6;&#x30A3;&#x3001;&#x30BB;&#x30EB;&#x8868;&#x9762;&#x9175;&#x7D20;&#x8907;&#x5408;&#x4F53;&#x3068;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x7DDA;&#x7DAD;&#x306E; 2 &#x3064;&#x306E;&#x69CB;&#x9020;&#x3067; ECM &#x57CB;&#x3081;&#x8FBC;&#x307E;&#x308C;&#x3066;&#x3067;&#x3059;&#x304B;&#xFF1F;&#x3053;&#x3053;&#x3067;&#x6211;&#x3005; &#x8907;&#x5408;&#x4F53;&#x306E;&#x3059;&#x3079;&#x3066;&#x306E;&#x30B3;&#x30F3;&#x30DD;&#x30FC;&#x30CD;&#x30F3;&#x30C8;&#x304C;&#x8868;&#x9762;&#x4E0A;&#x306E;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x7D30;&#x7DDA;&#x7DAD;&#x306E;&#x30D7;&#x30ED;&#x30BB;&#x30C3;&#x30B7;&#x30D6;&#x904B;&#x52D5;&#x304C;&#x53EF;&#x80FD;&#x3067;&#x3042;&#x308B;&#x3053;&#x3068;&#x3092;&#x793A;&#x3057;&#x307E;&#x3059;&#x3002;MT1 MMP &#x904B;&#x52D5;&#x306E;&#x6A5F;&#x69CB;&#x86CB;&#x767D;&#x8CEA;&#x5206;&#x89E3;&#x306B;&#x4F9D;&#x5B58;&#x3059;&#x308B;&#x30D0;&#x30A4;&#x30A2;&#x30B9;&#x6210;&#x5206;&#x3092;&#x504F;&#x308A;&#x306E;&#x3042;&#x308B;&#x62E1;&#x6563;&#x30A2;&#x30C7;&#x30CE;&#x30B7;&#x30F3;&#x4E09;&#x30EA;&#x30F3;&#x9178; &#xFF08;ATP&#xFF09; &#x306E;&#x52A0;&#x6C34;&#x5206;&#x89E3;&#x3067;&#x304D;&#x306A;&#x3044;&#x305D;&#x306E;&#x57FA;&#x677F;&#x306E;&#x3067;&#x3059;&#x3002;&#x3053;&#x308C;&#x306B;&#x4F3C;&#x3066;&#x3044;&#x308B; MMP 1 &#x30D6;&#x30E9;&#x30A6;&#x30F3;&#x904B;&#x52D5;&#x306E;&#x30E9;&#x30C1;&#x30A7;&#x30C3;&#x30C8;&#x3053;&#x3068;&#x6211;&#x3005; &#x306F;&#x524D;&#x306B;&#x8AAC;&#x660E;&#x3057;&#x307E;&#x3057;&#x305F;&#x3002;&#x52A0;&#x3048;&#x3066;&#x3001;&#x4E21;&#x65B9; MMP 2 &#x3068; MMP 9 &#x3068;&#x540C;&#x69D8;&#x30C1;&#x30F3;&#x30D7; 1 &#x53CA;&#x3073;-2&#x3001;&#x305D;&#x308C;&#x305E;&#x308C;&#x932F;&#x4F53; MMP 9 &#x306E;&#x30C0;&#x30A4;&#x30DE;&#x30FC;&#x5316;&#x9175;&#x7D20;&#x4E0D;&#x52D5;&#x30EC;&#x30F3;&#x30C0;&#x30EA;&#x30F3;&#x30B0;&#x3059;&#x308B;&#x9593;&#x306E;&#x9855;&#x8457;&#x306A;&#x89E3;&#x96E2;&#x306A;&#x3057;&#x306E;&#x30CD;&#x30A4;&#x30C6;&#x30A3;&#x30D6;&#x306E;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x7DDA;&#x7DAD;&#x306E;&#x8868;&#x9762;&#x306B;&#x30D6;&#x30E9;&#x30A6;&#x30F3;&#x62E1;&#x6563;&#x304C;&#x53EF;&#x80FD;&#x3067;&#x3059;&#x3002;&#x4E0D;&#x6D3B;&#x5316;&#x9175;&#x7D20;&#x6D3B;&#x6027; MT1 MMP &#x306E;&#x5C0F;&#x578B; 3 D &#x7D44;&#x7E54;&#x30B3;&#x30F3;&#x30B9;&#x30C8;&#x30E9;&#x30AF;&#x30C8;&#x3067;&#x958B;&#x767A;&#x3055;&#x308C;&#x305F;&#x30BB;&#x30EB;&#x529B;&#x3001;&#x691C;&#x51FA;&#x53EF;&#x80FD;&#x306A;&#x5426;&#x5B9A;&#x7684;&#x306A;&#x52B9;&#x679C;&#x304C;&#x3042;&#x308A;&#x307E;&#x3059;&#x3092;&#x898B;&#x3064;&#x3051;&#x308B;&#x6700;&#x3082;&#x6709;&#x76CA;&#x3067;&#x3059;&#x3002;&#x6211;&#x3005; &#x304C;&#x63D0;&#x6848;&#x3059;&#x308B;&#x30B3;&#x30E9;&#x30B2;&#x30CA;&#x30FC;&#x30BC;&#x8907;&#x96D1; &#xFF08;MT1 MMP&#xFF09; (2)/&#x30C1;&#x30F3;&#x30D7; 2/MMP 2 &#x30E2;&#x30D0;&#x30A4;&#x30EB; &#x30BB;&#x30EB;&#x8868;&#x9762;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x4E0B;&#x5730;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x3092;&#x8868;&#x3057;&#x307E;&#x3059;&#x3002;MT1 MMP MMP 2 &#x3068;&#x306E;&#x8907;&#x5408;&#x4F53;&#x306E;&#x7D30;&#x80DE;&#x8868;&#x9762;&#x306B;&#x3064;&#x306A;&#x304C;&#x308C;&#x305F;&#x5206;&#x5B50;&#x306E;&#x6B6F;&#x6B62;&#x3081;&#x3068;&#x3057;&#x3066;&#x6F14;&#x6280;&#x306E;&#x751F;&#x7269;&#x5B66;&#x7684;&#x542B;&#x610F;&#x30BB;&#x30EB; &#x30DE;&#x30C8;&#x30EA;&#x30C3;&#x30AF;&#x30B9;&#x306E;&#x76F8;&#x4E92;&#x4F5C;&#x7528;&#x306E;&#x7A7A;&#x9593;&#x7684;&#x898F;&#x5236;&#x306E;&#x30DA;&#x30EA;&#x643A;&#x5E2F;&#x30BF;&#x30F3;&#x30D1;&#x30AF;&#x8CEA;&#x5206;&#x89E3;&#x306E;&#x5F79;&#x5272;&#x306E;&#x305F;&#x3081;&#x306E;&#x65B0;&#x3057;&#x3044;&#x30E1;&#x30AB;&#x30CB;&#x30BA;&#x30E0;&#x3092;&#x63D0;&#x6848;&#x3057;&#x307E;&#x3059;&#x3002;

&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x7DDA;&#x7DAD;&#x306E;&#x8868;&#x9762;&#x306B; Mmp &#x306E;&#x62E1;&#x6563;: &#x30E2;&#x30D0;&#x30A4;&#x30EB;&#x643A;&#x5E2F;&#x8868;&#x9762;&#x30B3;&#x30E9;&#x30FC;&#x30B2;&#x30F3;&#x4E0B;&#x5730;&#x30A4;&#x30F3;&#x30BF;&#x30FC;&#x30D5;&#x30A7;&#x30A4;&#x30B9;&#x3002;

&#xAC1C;&#xBC1C; &#xD558;&#xB294; &#xB3D9;&#xC548; &#xC804;&#xCCB4;&#xC801; &#xD604;&#xC0C1;&#xC758; &#xC911;&#xC2EC;&#xC740; &#xAE30;&#xC9C8; MMPs &#xD558; &#xC5EC; &#xCD09;&#xB9E4;&#xC758; &#xB9AC; &#xBAA8;&#xB378;&#xB9C1; &#xBC0F; &#xC0C1;&#xCC98; &#xCE58;&#xC720;&#xB85C; &#xC12C;&#xC720; &#xC99D;, &#xC554; &#xB4F1; &#xC218;&#xB9CE;&#xC740; pathologic &#xC0C1;&#xD0DC;&#xC5D0;&#xC11C;. &#xC6B0;&#xB9AC;&#xB294; &#xC774;&#xC804;&#xC5D0; &#xBE44; MMP-2 &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xB2FF;&#xB294; &#xADF8;&#xB9AC;&#xACE0; MT1-MMP/TIMP-2-&#xC885;&#xC18D; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC5D0; &#xC758;&#xD574; &#xD65C;&#xC131;&#xD654; &#xC99D;&#xBA85; &#xD558;&#xACE0;&#xC788;&#xB2E4;. &#xACB0;&#xACFC; &#xC138;&#xD3EC; &#xD45C;&#xBA74; collagenolytic &#xBCF5;&#xC7A1; &#xD55C; (MT1 MMP) (2) / TIMP-2/MMP-2 (MT1 MMP)&#xB97C; &#xC2DC;&#xC791; &#xD558; &#xACE0; &#xAE30;&#xBCF8; &#xCF5C;&#xB77C;&#xAC90; fibril&#xC758; &#xC800;&#xD558; (MMP-2)&#xB97C; &#xC644;&#xB8CC; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4;. &#xB2E4;&#xC74C;&#xACFC; &#xAC19;&#xC740; &#xC9C8;&#xBB38;&#xC774; &#xB0A8;&#xC544;: ECM&#xC5D0; &#xD3EC;&#xD568; &#xB41C; &#xC0C1;&#xB300;&#xC801;&#xC73C;&#xB85C; &#xC81C;&#xD55C; &#xB41C; &#xC774;&#xB3D9;&#xC131;, &#xC140; &#xD45C;&#xBA74; &#xD6A8;&#xC18C; &#xBCF5;&#xD569;&#xBB3C;&#xACFC; &#xCF5C;&#xB77C;&#xAC90; fibril &#xB450; &#xAD6C;&#xC870; &#xAE30;&#xD310; &#xC778;&#xC2DD; &#xAD00;&#xB828; &#xB41C; &#xBA54;&#xCEE4;&#xB2C8;&#xC998; &#xC774;&#xB780; &#xBB34;&#xC5C7;&#xC785;&#xB2C8;&#xAE4C;? &#xC5EC;&#xAE30; &#xC6B0;&#xB9AC;&#xB294; &#xB2E8;&#xC9C0;&#xC758; &#xBAA8;&#xB4E0; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB294; &#xCF5C;&#xB77C;&#xAC90; fibril&#xC758; &#xD45C;&#xBA74;&#xC5D0; processive &#xC6B4;&#xB3D9;&#xC758; &#xB2A5;&#xB825;&#xC744; &#xBCF4;&#xC5EC;&#xC90D;&#xB2C8;&#xB2E4;. MT1 MMP &#xC6B4;&#xB3D9;&#xC758; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC740; &#xD558;&#xC9C0; &#xC544;&#xB370;&#xB178;&#xC2E0; &#xC0BC;&#xC778;&#xC0B0; (ATP)&#xC758; &#xAC00;&#xC218;&#xBD84;&#xD574;&#xB294; &#xAE30;&#xD310; &#xBC14;&#xC774;&#xC5B4;&#xC2A4; &#xAD6C;&#xC131; &#xC694;&#xC18C;&#xB294; &#xBCA0;&#xC774;&#xC2A4;&#xC5D0; &#xC758;&#xC874; &#xD3B8;&#xACAC;&#xB41C; &#xD655;&#xC0B0;. &#xADF8;&#xAC83;&#xC740; &#xBE44;&#xC2B7;&#xD55C; &#xC55E;&#xC5D0;&#xC11C; &#xC124;&#xBA85;&#xD55C; &#xADF8;&#xC758; MMP 1 Brownian &#xB798;&#xCE58; &#xC6B0;&#xB9AC;. &#xB610;&#xD55C;, &#xBAA8;&#xB450; MMP 2 &#xBC0F; MMP 9 &#xBFD0;&#xB9CC; &#xC544;&#xB2C8;&#xB77C; TIMP 1&#xACFC;-2 &#xB2E8;&#xC9C0; &#xADF8;&#xB4E4;&#xC758; &#xC218; &#xC788;&#xC2B5;&#xB2C8;&#xB2E4; &#xB208;&#xC5D0; &#xB744;&#xB294; &#xBD84;&#xB9AC; &#xC5C6;&#xC774; &#xB124;&#xC774;&#xD2F0;&#xBE0C; &#xCF5C;&#xB77C;&#xAC90; &#xC18C;&#xC758; &#xD45C;&#xBA74;&#xC5D0; Brownian &#xBCF4;&#xAE09;&#xC758; MMP 9 dimerization &#xC6C0;&#xC9C1;&#xC774;&#xAE30; &#xD798; &#xB4DC;&#xB294; &#xD6A8;&#xC18C;&#xB97C; &#xB80C;&#xB354;&#xB9C1; &#xD558;&#xB294; &#xB3D9;&#xC548;. &#xAC00;&#xC7A5; &#xC720;&#xC775;&#xD55C; MT1 MMP &#xD6A8;&#xC18C; &#xD65C;&#xB3D9;&#xC758; &#xBE44;&#xD65C;&#xC131;&#xD654;&#xB294; &#xC18C;&#xD615;&#xB41C; 3D &#xC870;&#xC9C1; &#xAD6C;&#xC870;&#xC5D0;&#xC11C; &#xAC1C;&#xBC1C;&#xD55C; &#xC140; &#xD3EC;&#xC2A4;&#xC5D0; &#xBD80;&#xC815;&#xC801;&#xC778; &#xC601;&#xD5A5;&#xC744; &#xAC10;&#xC9C0;&#xD560; &#xC218; &#xC788;&#xB294; &#xBC1C;&#xACAC;&#xC774; &#xC774;&#xB2E4;. &#xC6B0;&#xB9AC;&#xAC00; &#xC81C;&#xC548; &#xD558;&#xB294; collagenolytic &#xBCF5;&#xD569; (MT1 MMP) (2) / TIMP-2/MMP-2 &#xBAA8;&#xBC14;&#xC77C; &#xC140; &#xD45C;&#xBA74; &#xCF5C;&#xB77C;&#xAC90; &#xD558; &#xCE35; &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;&#xB97C; &#xB098;&#xD0C0;&#xB0C5;&#xB2C8;&#xB2E4;. MT1 MMP MMP 2 &#xBCF5;&#xC7A1; &#xD55C; &#xC138;&#xD3EC; &#xD45C;&#xBA74;&#xC5D0; &#xB2FF;&#xB294; &#xBD84;&#xC790; &#xB798; &#xCE6B; &#xC5ED;&#xD560;&#xC758; &#xC0DD;&#xBB3C; &#xD559;&#xC801; &#xC601;&#xD5A5; &#xC138;&#xD3EC;-&#xB9E4;&#xD2B8;&#xB9AD;&#xC2A4; &#xC0C1;&#xD638; &#xC791;&#xC6A9;&#xC5D0;&#xC11C; &#xACF5;&#xAC04;&#xC801;&#xC73C;&#xB85C; &#xB808; &#xADE4; &#xB808;&#xC774;&#xD2B8; &#xB41C; peri &#xC140;&#xB8F0;&#xB7EC; &#xBCA0;&#xC774;&#xC2A4;&#xC758; &#xC5ED;&#xD560;&#xC5D0; &#xB300; &#xD55C; &#xC0C8;&#xB85C;&#xC6B4; &#xBA54;&#xCEE4;&#xB2C8;&#xC998;&#xC744; &#xC81C;&#xC548; &#xD569;&#xB2C8;&#xB2E4;.

&#xCF5C;&#xB77C;&#xAC90; &#xC18C;&#xC758; &#xD45C;&#xBA74;&#xC5D0; Mmps&#xC758; &#xBCF4;&#xAE09;: &#xBAA8;&#xBC14;&#xC77C; &#xC140; &#xD45C;&#xBA74; &#xCF5C;&#xB77C;&#xAC90; &#xD558; &#xCE35; &#xC778;&#xD130;&#xD398;&#xC774;&#xC2A4;.

Remodelaci&oacute;n de la matriz extracelular catalizada por MMPs es fundamental para fen&oacute;menos morfogen&eacute;ticos durante el desarrollo y la herida curativa as&iacute; como en numerosos estados patol&oacute;gicos tales como fibrosis y c&aacute;ncer. Hemos demostrado previamente que secretada MMP-2 est&aacute; anclado a la superficie celular y activada por un mecanismo dependiente de MT1-MMP/TIMP-2. La resultante colagenol&iacute;tica de superficie celular compleja (MT1-MMP) 2/TIMP-2/MMP-2 puede iniciar (MT1-MMP) y completa degradaci&oacute;n (MMP-2) de una fibrilla de col&aacute;geno subyacente. La siguiente cuesti&oacute;n: &iquest;Cu&aacute;l es el mecanismo de reconocimiento de sustrato que implican las dos estructuras de movilidad relativamente restringido, el complejo enzim&aacute;tico superficial de la c&eacute;lula y una fibrilla de col&aacute;geno incrustado en el ECM? Aqu&iacute; demostramos que todos los componentes del complejo son capaces de movimiento procesual sobre una superficie de la fibrilla de col&aacute;geno. El mecanismo de movimiento de MT1-MMP es una difusi&oacute;n parcial con el componente diagonal depende de la proteolisis de su sustrato, no hidr&oacute;lisis de trifosfato de adenosina (ATP). Es similar a que de la MMP-1 browniano trinquete se ha descrito anteriormente. Adem&aacute;s, tanto MMP-2 y MMP-9, as&iacute; como sus respectivos complejos con TIMP-1 y -2 son capaces de difusi&oacute;n browniana en la superficie de las fibrillas de col&aacute;geno nativo sin disociaci&oacute;n sensible mientras la dimerizaci&oacute;n de MMP-9 representa la enzima inm&oacute;vil. M&aacute;s ilustrativo es la conclusi&oacute;n que la inactivaci&oacute;n de la actividad enzim&aacute;tica de MT1-MMP tiene un efecto negativo detectable en la fuerza de la c&eacute;lula que se convirti&oacute; en tejido 3D miniaturizados construcciones. Proponemos que el colagenol&iacute;tica complejo (MT1-MMP) 2/TIMP-2/MMP-2 representa una interfaz de sustrato m&oacute;vil celular superficie-col&aacute;geno. Las implicaciones biol&oacute;gicas de MT1-MMP, actuando como un trinquete molecular anclado a la superficie de la c&eacute;lula en complejo con MMP-2 sugieren un nuevo mecanismo para el papel de la prote&oacute;lisis de peri-celular espacialmente regulado en interacciones c&eacute;lula-matriz.

Difusi&oacute;n de MMP en la superficie de las fibrillas de col&aacute;geno: la interfaz de superficie-col&aacute;geno sustrato m&oacute;vil celular.

Vesicular stomatitis virus (VSV) is a prototypic negative sense single-stranded RNA virus. The bullet-shape appearance of the virion results from tightly wound helical turns of the nucleoprotein encapsidated RNA template (N-RNA) around a central cavity. Transcription and replication require polymerase complexes, which include a catalytic subunit L and a template-binding subunit P. L and P are inferred to be in the cavity, however lacking direct observation, their exact position has remained unclear. Using super-resolution fluorescence imaging and atomic force microscopy (AFM) on single VSV virions, we show that L and P are packaged asymmetrically towards the blunt end of the virus. The number of L and P proteins varies between individual virions and they occupy 57 ± 12 nm of the 150 nm central cavity of the virus. Our finding positions the polymerases at the opposite end of the genome with respect to the only transcriptional promoter.

Asymmetric packaging of polymerases within vesicular stomatitis virus.

HIV Gag polymerizes on the plasma membrane to form virus like particles (VLPs) that have similar diameters to wild-type viruses. We use multicolor, dual-penetration depth, total internal reflection fluorescence microscopy, which corrects for azimuthal movement, to image the assembly of individual VLPs from the time of nucleation to the recruitment of VPS4 (a component of the endosomal sorting complexes required for transport, and which marks the final stage of VLP assembly). Using a mathematical model for assembly and maximum-likelihood comparison of fits both with and without pauses, we detect pauses during Gag polymerization in 60% of VLPs. Pauses range from 2 to 20 min, with an exponentially distributed duration that is independent of cytosolic Gag concentration. VLPs assembled with late domain mutants of Gag (which do not recruit the key endosomal sorting complexes required for transport proteins Alix or TSG101) exhibit similar pause distributions. These pauses indicate that a single rate-limiting event is required for continuation of assembly. We suggest that pauses are either related to incorporation of defects in the hexagonal Gag lattice during VLP assembly or are caused by shortcomings in interactions of Gag with essential and still undefined cellular components during formation of curvature on the plasma membrane.

Saveez Saffarian

Department of Physics & Astronomy,
Center for Cell and Genome

University of Utah

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Saveez Saffarian

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Physics & Astronomy,Center for Cell and Genome

University of Utah

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Department of Physics & Astronomy,
Center for Cell and Genome