Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients

Irimia, Daniel; Liu, Su-Yang; Tharp, William G.; Samadani, Azadeh; Toner, Mehmet; Poznansky, Mark C.

doi:10.1039/b511877h

Cited by 167 publications

(177 citation statements)

References 29 publications

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“…For example, the model predicts that when the new external cue is aligned with the internal memory it may establish polarization more rapidly than when an external cue is oriented in opposition to the internal memory. Further, kinetic measurements would begin to dissect interprocess time scales and explain behaviors such as U-turns originally described by Zigmond et al (33) that occur at slow changes in the external environment but disappear in rapidly changing gradients (34) and maintain polarity in the face of reversed chemoattractant gradients (35). More generally, our study shows how the temporal separation of membrane and cytoskeletal polarizations allows a cell to sense and respond to a fluctuating environment robustly.…”

Section: Discussionmentioning

confidence: 82%

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Prentice-Mott

Meroz

Carlson

et al. 2016

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

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Chemotaxis, the directional migration of cells in a chemical gradient, is robust to fluctuations associated with low chemical concentrations and dynamically changing gradients as well as high saturating chemical concentrations. Although a number of reports have identified cellular behavior consistent with a directional memory that could account for behavior in these complex environments, the quantitative and molecular details of such a memory process remain unknown. Using microfluidics to confine cellular motion to a 1D channel and control chemoattractant exposure, we observed directional memory in chemotactic neutrophil-like cells. We modeled this directional memory as a long-lived intracellular asymmetry that decays slower than observed membrane phospholipid signaling. Measurements of intracellular dynamics revealed that moesin at the cell rear is a longlived element that when inhibited, results in a reduction of memory. Inhibition of ROCK (Rho-associated protein kinase), downstream of RhoA (Ras homolog gene family, member A), stabilized moesin and directional memory while depolymerization of microtubules (MTs) disoriented moesin deposition and also reduced directional memory. Our study reveals that long-lived polarized cytoskeletal structures, specifically moesin, actomyosin, and MTs, provide a directional memory in neutrophil-like cells even as they respond on short time scales to external chemical cues. . In particular, chemical cues activate signaling at the cell surface and are integrated within the cell cytoplasm to give rise to a polarization in the direction of the local gradient. Although these processes have been the subject of detailed experimental study and theoretical and computational modeling, the mechanisms by which this orientation is achieved and maintained in the face of environmental noise remains incomplete.Although migration might be thought of as being very sensitive to the variations in the external environment, instead, we see robust migration in a variety of fluctuating environments. These observations all point to the existence of a directional memory in chemotactic cells-a biochemical pathway that stores information about cellular orientation and prevents the loss of orientation in the face of fluctuations, transient loss of polarization, or saturation of the receptors. For example, recent work has demonstrated memory-based behavior in Dictyostelium amoebae under fluctuating waves of chemoattractant (6, 7), although the authors do not identify potential molecular elements that store this information.Here, we use microchannel-based microfluidic devices to observe cell polarization and movement in confined mammalian neutrophil-like cells. Cells in this environment exhibit a strong bias to repolarize in the previous direction of motion after a period of depolarization. This memory is time-dependent and decays when the cell is unstimulated. To describe these results, we construct a minimal phenomenological model coupling membrane and cytoskeletal polarization lifetimes and show that thi...

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Section: Discussionmentioning

confidence: 82%

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Prentice-Mott

Meroz

Carlson

et al. 2016

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

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“…However, the number of required inputs would increase with the number of inflection points leading to more complex designs for the universal gradient devices. Finally, while the present device has been designed for steady concentration profiles, temporal variations of the gradients would be possible by the integration of microstructured valves in the microfluidic design, 19 with applications such as the study of the initiation of neutrophil migration 19 or axonal growth or regeneration. 20 …”

Section: Resultsmentioning

confidence: 99%

Universal Microfluidic Gradient Generator

2006

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The study of cellular responses to chemical gradients in vitro would greatly benefit from experimental systems that can generate precise and stable gradients comparable to chemical nonhomogeneities occurring in vivo. Recently, microfluidic devices have been demonstrated for linear gradient generation for biological applications with unmatched accuracy and stability. However, no systematic approach exists at this time for generating other gradients of target spatial configuration. Here we demonstrate experimentally and provide mathematical proof for a systematic approach to generating stable gradients of any profile by the controlled mixing of two starting solutions.Replication of complex chemical gradients is essential for many in vitro experimental studies in prokaryotic and eukaryotic cells. Investigation of fundamental processes such as the migration of prokaryotic cell toward sources of nutrients, 1 immune cells against intruders, 2,3 the growth or regeneration of axons toward their connection target, 4 and the differentiation of embryonic cells in response to morphogens 5 require precise duplication of the in vivo spatial distribution of various signaling molecules. Such distributions usually occur in nonhomogeneous mediums, under various production, transport, and degradation conditions, and gradients of nonlinear, more or less complex shapes are usually formed. 6,7 Although linear gradients represent a good first-order approximation for in vivo gradients, and are useful in gaining insights into the biology of cellular responses, growing evidence suggests that a lot of complexity arises and many cellular responses are specific to spatial gradients that are not linear. For example, in bacteria migrating in chemoattractant gradients, the interplay between sensory adaptation and concentration changes due to displacement can lead to variations in chemotactic responses. 8 Similarly, cancerous cells that typically do not respond to linear chemotactic gradients have been recently shown to migrate in response to nonlinear gradients. 9 As a result, it is critical for better understanding of cellular polarization, migration, growth, or division responses of cells to biochemical heterogeneities in their microenvironment to be able to consistently replicate chemical concentration profiles comparable to those experienced by cells in vivo. Still, most of the current experimental systems for generating chemical gradients, e.g., Boyden chambers, 10 Dunn chambers, 11 or microfluidic gradient generators, 12 only produce linear gradients of soluble biochemical factors, and few alternatives exist for producing gradients of other spatial profiles at scales comparable to cell size. While some gradients that are not linear have been generated using asymmetric flow in symmetric networks, 13 or serial dilutions schemes, 14,15

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“…This behavior is seen when chemoattractant gradients are presented at the rear of polarized cells through microfluidics or a micropipette. Chemoattractant-induced repolarization can be seen in neutrophils (48), social amoebae (49), and breast cancer cells (36). Given that rapa- mycin-induced graded Rac activation can mimic polarization behaviors seen with chemoattractant gradients, we believe that polarization can be defined at the level of Rac, or at least starting from the level of Rac.…”

Section: Discussionmentioning

confidence: 99%

Synthetic spatially graded Rac activation drives cell polarization and movement

Lin

Holmes

Wang

et al. 2012

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

101

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Migrating cells possess intracellular gradients of active Rho GTPases, which serve as central hubs in transducing signals from extracellular receptors to cytoskeletal and adhesive machinery. However, it is unknown whether shallow exogenously induced intracellular gradients of Rho GTPases are sufficient to drive cell polarity and motility. Here, we use microfluidic control to generate gradients of a small molecule and thereby directly induce linear gradients of active, endogenous Rac without activation of chemotactic receptors. Gradients as low as 15% were sufficient not only to trigger cell migration up the chemical gradient but to induce both cell polarization and repolarization. Cellular response times were inversely proportional to the steepness of Rac inducer gradient in agreement with a mathematical model, suggesting a function for chemoattractant gradient amplification upstream of Rac. Increases in activated Rac levels beyond a well-defined threshold augmented polarization and decreased sensitivity to the imposed gradient. The threshold was governed by initial cell polarity and PI3K activity, supporting a role for both in defining responsiveness to Rac activation. Our results reveal that Rac can serve as a starting point in defining cell polarity. Furthermore, our methodology may serve as a template to investigate processes regulated by intracellular signaling gradients.cell signaling | synthetic biology | chemotaxis | signal transduction

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Microfluidic system for measuring neutrophil migratory responses to fast switches of chemical gradients

Cited by 167 publications

References 29 publications

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Directional memory arises from long-lived cytoskeletal asymmetries in polarized chemotactic cells

Universal Microfluidic Gradient Generator

Synthetic spatially graded Rac activation drives cell polarization and movement

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