Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

Lin, Hai; Clegg, Dennis O.; Lal, Ratnesh

doi:10.1021/bi990800q

Cited by 61 publications

(51 citation statements)

References 22 publications

(35 reference statements)

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“…In addition, previous experiments have been reported on the cleavage of native and site-directed mutants of murine collagen by MMP-1, outlining crucial aspects of the recognition mechanism (17). Very recently, proteolysis of single collagen I molecules has been followed by atomic force microscopy (18), confirming that the process follows a simple Michaelis-Menten mechanism and outlining that the results on bulk solution are compatible with those observed on single collagen molecules.…”

Section: Matrix Metalloproteinases (Mmp)supporting

confidence: 56%

Cleavage of Bovine Collagen I by Neutrophil Collagenase MMP-8

Marini¹,

Fasciglione²,

Sanctis³

et al. 2000

Journal of Biological Chemistry

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The enzymatic processing of bovine collagen I by neutrophil collagenase (MMP-8) has been monitored at 37°C, envisaging the occurrence of multiple intermediate steps, following the initial cleavage, which leads to the formation of 1 ⁄4 and 3 ⁄4 fragments. Further, the first cleavage event has been investigated at 37°C as a function of pH, and catalytic parameters have been obtained through a global analysis of steady-state kinetic data, such as to get an overall consistent picture of k cat /K m , k cat , and K m . These data have been compared with those obtained from the catalysis by MMP-8 of two synthetic fluorogenic substrates under the same experimental conditions. The overall behavior can be accounted for by the existence of five protonating groups, which vary to a different extent their pK a values for the three substrates investigated. The main observation concerns the fact the two of these residues, which play a relevant role in the enzymatic activity of MMP-8, are relatively far from the primary recognition site, and they are coming into action only for large macromolecular substrates, such as bovine collagen I. This finding opens the question of appropriate testing for inhibitors of the enzymatic action of MMP-8, which must take into account, and also of these relevant interactions occurring only with natural substrates. Matrix metalloproteinases (MMP)1 are a class of proteolytic enzymes characterized by the presence of a Zn 2ϩ atom in the active site, which are able to process enzymatically several components of the extracellular matrix, such as interstitial and basement membranes, collagen, fibronectin, and laminin. Because of their crucial role in the extracellular matrix degradation, they are implicated in the tissue remodeling processes associated to the growth and development and in several pathological conditions, such as rheumatoid arthritis and tumor invasion (1-3). Collagenases, such as fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), and collagenase-3 (MMP-13), are a class of MMPs that specifically cleave several types of native triple-helical collagen (4 -6), and in particular collagen I, at a specific peptide bond between Gly 775 and the residue in position 776 (which can be either Leu or Ile) (4, 7), leading to the formation of 1 ⁄4 and 3 ⁄4 fragments. This event is a crucial one because of the fairly slow rate of collagen turnover within the cartilage (8), and it is closely related to the occurrence of important physiological and pathological events (9 -11).Collagen I is one of the major constituents of the extracellular matrix for several tissues, such as skin, tendon, blood vessels, cartilage, bones, and basal laminas (12), and its triplehelical structural arrangement has been the object of several recent studies both on the native molecule (Ref. 13 and references quoted therein) and on model peptides (14, 15). The investigation on the action of MMPs on collagen has been carried out for a long time, and preliminary measurements have been carried out on the activation ener...

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Section: Matrix Metalloproteinases (Mmp)supporting

confidence: 56%

Cleavage of Bovine Collagen I by Neutrophil Collagenase MMP-8

Marini¹,

Fasciglione²,

Sanctis³

et al. 2000

Journal of Biological Chemistry

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“…These two groups continued these studies and obtained time-lapse images (~30 s intervals) of the RNA transcription reaction between DNA and RNA polymerase [42] and of the 1D diffusion of RNA polymerase along a DNA strand [43]. Other examples of dynamic processes that have been imaged are the proteolytic cleavage of collagen I by collagenase [44] and nuclear-pore closing by exposure to CO 2 [45].…”

Section: History Of Afm Studies On Biomolecular Processesmentioning

confidence: 98%

High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes

Ando

Uchihashi

Fukuma

2008

Progress in Surface Science

492

414

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The atomic force microscope (AFM) has a unique capability of allowing the high-resolution imaging of biological samples on substratum surfaces in physiological solutions. Recent technological progress of AFM in biological research has resulted in remarkable improvements in both the imaging rate and the tip force acting on the sample. These improvements have enabled the direct visualization of dynamic structural changes and dynamic interactions occurring in individual biological macromolecules, which is currently not possible with other techniques. Therefore, high-speed AFM is expected to have a revolutionary impact on biological sciences. In addition, the recently achieved atomic resolution in liquids will further expand the usefulness of AFM in biological research. In this article, we first describe the various capabilities required of AFM in biological sciences, which is followed by a detailed description of various devices and techniques developed for high-speed AFM and atomic-resolution in-liquid AFM. We then describe various imaging studies performed using our cutting-edge microscopes and their current capabilities as well as their limitations, and conclude by discussing the future prospects of AFM as an imaging tool in biological research.

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“…Imaging of the cleavage of tropocollagen II by MMP-8 with single-molecule resolution by AFM revealed the existence of several MMP-8-binding sites on the tropocollagen molecules, although cleavage occurs only in one specific location [82].…”

Section: Cellular Microecology and Proteolytic Activitymentioning

confidence: 99%

Probing cellular microenvironments and tissue remodeling by atomic force microscopy

Ludwig

Kirmse

Poole

et al. 2007

Pflugers Arch - Eur J Physiol

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The function of cells is strongly determined by the properties of their extracellular microenvironment. Biophysical parameters like environmental stiffness and fiber orientation in the surrounding matrix are important determinants of cell adhesion and migration. Processes like tissue maintenance, wound repair, cancer cell invasion, and morphogenesis depend critically on the ability of cells to actively sense and remodel their surroundings. Pericellular proteolytic activity and adaptation of migration tactics to the environment are strategies to achieve this aim. Little is known about the distinct regulatory mechanisms that are involved in these processes. The system's critical biophysical and biochemical determinants are well accessible by atomic force microscopy (AFM), a unique tool for functional, nanoscale probing and morphometric, highresolution imaging of processes in live cells. This review highlights common principles of tissue remodeling and focuses on application examples of different AFM techniques, for example elasticity mapping, the combination of AFM and fluorescence microscopy, the morphometric imaging of proteolytic activity, and force spectroscopy applications of single molecules or individual cells. To achieve a more complete understanding of the processes underlying the interaction of cells with their environments, the combination of AFM force spectroscopy experiments will be essential.

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Imaging Real-Time Proteolysis of Single Collagen I Molecules with an Atomic Force Microscope

Cited by 61 publications

References 22 publications

Cleavage of Bovine Collagen I by Neutrophil Collagenase MMP-8

Cleavage of Bovine Collagen I by Neutrophil Collagenase MMP-8

High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes

Probing cellular microenvironments and tissue remodeling by atomic force microscopy

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