New insights into extracellular matrix assembly and reorganization from dynamic imaging of extracellular matrix proteins in living osteoblasts

Pitchumani Sivakumar,Sarah L Dallas,Vivek P Divakara,Yu-Ping Wang,Andras Czirok,Brenda J Rongish

doi:10.1242/jcs.02830

Pitchumani Sivakumar, Sarah L Dallas + Show 4 more

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https://doi.org/10.1242/jcs.02830

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Abstract

The extracellular matrix (ECM) has been traditionally viewed as a static scaffold that supports cells and tissues. However, recent dynamic imaging studies suggest that ECM components are highly elastic and undergo continual movement and deformation. Latent transforming growth factor beta (TGFbeta) binding protein-1 (LTBP1) is an ECM glycoprotein that binds latent TGFbeta and regulates its availability and activity. LTBP1 initially co-distributes with fibronectin in the extracellular matrix of osteoblasts, and depends on fibronectin for its assembly. To gain further insights into the mechanisms of assembly of LTBP1 and its spatial and temporal interactions with fibronectin, we have performed dual fluorescence time-lapse imaging of these two proteins in living osteoblasts using fluorescent probes. Time-lapse movies showed surprisingly large fibril displacements associated with cellular movement as well as occasional breaking of LTBP1 or fibronectin-containing fibrils. Individual fibrils stretched to as much as 3.5 times or contracted to as much as one fourth of their original length. Motile cells appeared to actively mediate extracellular matrix assembly by adding 'globules' or 'packets' of matrix material onto existing fibrils. They also actively reorganized the extracellular matrix by shunting matrix material from one location to another and exchanging fibrillar material between fibrils. This cell-mediated matrix reorganization was primarily associated with the assembly and remodeling of the initial (early) matrix, whereas mature, established ECM was more stable. Displacement vector mapping showed that different matrix fibrillar networks within the same cultures can show different dynamic motion in response to cell movement and showed that the motion of fibrils was correlated with cell motion. These data suggest novel cell-mediated mechanisms for assembly and reorganization of the extracellular matrix and highlight a role for cell motility in the assembly process.

Highlights

The extracellular matrix (ECM) controls cell function by regulating cell-matrix interactions, modulating extracellular signals and providing a storage site for growth factors and cytokines
Displacement vector mapping showed that different matrix fibrillar networks within the same cultures can show different dynamic motion in response to cell movement and showed that the motion of fibrils was correlated with cell motion
We have shown that latent TGF␤ binding protein-1 (LTBP1) assembly is severely impaired in fibronectin-null embryonic fibroblasts and can be rescued by addition of exogenous fibronectin, suggesting that interactions between fibronectin and LTBP1 are critical for LTBP1 incorporation into the ECM (Dallas et al, 2005)

Summary

Introduction

The extracellular matrix (ECM) controls cell function by regulating cell-matrix interactions, modulating extracellular signals and providing a storage site for growth factors and cytokines. Type I collagen is the major component, the mineralized ECM of bone is composed of several noncollagenous proteins with distinct functions (reviewed by Young, 2003). Several studies have suggested that fibronectin is required for assembly of multiple ECM proteins, including collagen types I and III (McDonald et al, 1982; Velling et al, 2002), fibulin (Godyna et al, 1995; Roman and McDonald, 1993), fibrinogen (Pereira et al, 2002) and thrombospondin (Sottile and Hocking, 2002). We have previously shown that fibronectin is essential for assembly of latent TGF␤ binding protein-1 (LTBP1) into the ECM of bone cells (Dallas et al, 2005)

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