Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy

Wiseman, Paul W.; Brown, Claire M.; Webb, Donna J.; Hébert, Benedict; Johnson, Noor; Squier, Jeff; Ellisman, Mark H.; Horwitz, Alan F.

doi:10.1242/jcs.01416

Cited by 214 publications

(221 citation statements)

References 24 publications

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“…5A,B). Temporal image correlation spectroscopy analysis shows that >80% of the integrins are stationary relative to the substrate (Table 1), in accord with our earlier image correlation and FRAP measurements (Wiseman et al, 2004).…”

Section: The Integrin-actin Linkage In Cho Cellssupporting

confidence: 88%

“…CHO cells plated under these conditions do not show large organized adhesions in active protrusions, or prominent actin stress fibers. However, we have previously shown that, even under these conditions, the integrin is clustered and organized in immobile structures that are not visible by eye (Wiseman et al, 2004).…”

Section: The Integrin-actin Linkage In Cho Cellsmentioning

confidence: 85%

See 1 more Smart Citation

Probing the integrin-actin linkage using high-resolution protein velocity mapping

Brown

Hébert

Kolin

et al. 2006

Journal of Cell Science

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162

166

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Cell migration is regulated in part by the connection between the substratum and the actin cytoskeleton. However, the very large number of proteins involved in this linkage and their complex network of interactions make it difficult to assess their role in cell migration. We apply a novel image analysis tool, spatio-temporal image correlation spectroscopy (STICS), to quantify the directed movements of adhesion-related proteins and actin in protrusions of migrating cells. The STICS technique reveals protein dynamics even when protein densities are very low or very high, and works in the presence of large, static molecular complexes. Detailed protein velocity maps for actin and the adhesion-related proteins α-actinin, α5-integrin, talin, paxillin, vinculin and focal adhesion kinase are presented. The data show that there are differences in the efficiency of the linkage between integrin and actin among different cell types and on the same cell type grown on different substrate densities. We identify potential mechanisms that regulate efficiency of the linkage, or clutch, and identify two likely points of disconnect, one at the integrin and the other at α-actinin or actin. The data suggests that the efficiency of the linkage increases as actin and adhesions become more organized showing the importance of factors that regulate the efficiency in adhesion signaling and dynamics.

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Section: The Integrin-actin Linkage In Cho Cellssupporting

confidence: 88%

Section: The Integrin-actin Linkage In Cho Cellsmentioning

confidence: 85%

Probing the integrin-actin linkage using high-resolution protein velocity mapping

Brown

Hébert

Kolin

et al. 2006

Journal of Cell Science

Self Cite

162

166

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“…These original implementations of ICS did not include a temporal component. More recently, Srivastava and Petersen (27) and Wiseman et al (28,29) have described combined temporal and spatial autocorrelations. Although these studies have focused on model systems such as beads, the authors suggested that the method can be extended to analyze cytoplasmic dynamics.…”

mentioning

confidence: 99%

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

Kulkarni

Davis

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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Quantitatively understanding how nonviral gene delivery vectors (polyplexes) are transported inside cells is essential before they can be optimized for gene therapy and medical applications. In this study, we used spatio-temporal image correlation spectroscopy (ICS) to follow polymer-nucleic acid particles (polyplexes) of various sizes and analyze their diffusive-like and flow behaviors intracellularly to elucidate the mechanisms responsible for their transport. ICS is a quantitative imaging technique that allows the assessment of particle motion in complex systems, although it has not been widely used to date. We find that the internalized polyplexes are able to use microtubule motors for intracellular trafficking and exhibit different transport behaviors for short (<10 s) versus long (Ϸ60 s) correlation times. This motion can be explained by a memory effect of the microtubule motors. These results reveal that, although microtubule motor biases may be present for short periods of time, resulting in a net directional velocity, the overall long-term motion of the polyplexes is best described as a random walk-like process. These studies suggest that spatio-temporal ICS is a powerful technique for assessing the nature of intracellular motion and provides a quantitative tool to compare the transport of different objects within a living cell.transfection agent ͉ molecular mobility ͉ cytoplasmic crowding ͉ cyclodextrin R ecently, significant progress has been made in developing alternatives to viral-based gene therapy, motivated by safety concerns of viral infection. Understanding such nonviral gene delivery (one type of vector that involves the combination of synthetic polymers and nucleic acids to form particles called polyplexes and is used here) requires knowledge of how they behave within cells, including the mechanisms involved in polyplex trafficking such as endocytosis, cytoplasmic transport, endosomal escape, and nuclear localization (1-3). Recent reports have indicated the presence of significant bottlenecks in the delivery process, especially problems with endosomal escape (4). Measuring these dynamics, especially transport parameters, is important for understanding both how such delivery methods compare with viruses and how to improve their efficiencies.Confocal microscopy allows for good visualization of small quantities of fluorescently labeled species. However, only a few studies of cytoplasmic transport have focused on quantitative biophysical parameters such as effective diffusion constants or transport velocities (4-6). Most experiments measuring these parameters have focused on examining small injected oligonucleotides or proteins (7-11). In contrast, polyplexes that range in size from 75 to 250 nm or larger enter cells by endocytosis and become enclosed in endosomes (12). For transport of large entities (Ͼ30 nm), such as the polyplexes used for gene therapy, issues such as crowding and microtubule transport become critical (13,14).Here, we describe spatio-temporal image correlation spectroscopy (IC...

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“…This phenomenon is known as retrograde flow. There is a balance between the actin cytoskeleton assembly and the retrograde flow that govern and control cell movement [1,27,54,20].…”

Section: Structural Components Of Cell-matrix Adhesionmentioning

confidence: 99%

Intracellular Modelling of Cell-Matrix Adhesion during Cancer Cell Invasion

Andasari

Chaplain

2012

Math. Model. Nat. Phenom.

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Abstract. When invading the tissue, malignant tumour cells (i.e. cancer cells) need to detach from neighbouring cells, degrade the basement membrane, and migrate through the extracellular matrix. These processes require loss of cell-cell adhesion and enhancement of cell-matrix adhesion. In this paper we present a mathematical model of an intracellular pathway for the interactions between a cancer cell and the extracellular matrix. Cancer cells use similar mechanisms as with normal cells for their interactions with the extracellular matrix. We develop a model of cell-matrix adhesion that accounts for reactions between the cell surface receptor integrins, the matrix glycoprotein fibronectin, and the actin filaments in the cytoskeleton. Each represents components for an intermediate compartment, the extracellular compartment, and the intracellular compartment, respectively. Binding of fibronectin with integrins triggers a clustering of protein complexes, which then activates and phosphorylates regulatory proteins that are involved in actin reorganisation causing actin polymerization and stress fibre assembly. Rearrangement of actin filaments with integrin/fibronectin complexes near adhesion sites and interaction with fibrillar fibronectin produces the force necessary for cell migration, accounting for cell-matrix adhesion.

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Spatial mapping of integrin interactions and dynamics during cell migration by Image Correlation Microscopy

Cited by 214 publications

References 24 publications

Probing the integrin-actin linkage using high-resolution protein velocity mapping

Probing the integrin-actin linkage using high-resolution protein velocity mapping

Quantitating intracellular transport of polyplexes by spatio-temporal image correlation spectroscopy

Intracellular Modelling of Cell-Matrix Adhesion during Cancer Cell Invasion

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