u-track 3D: measuring and interrogating dense particle dynamics in three dimensions

Roudot, Philippe; Wr, Legant; Zou, Quan; Km, Dean; Es, Welf; David, Ana F.; Gerlich, Daniel W.; Rp, Fiolka; Betzig, Eric; Danuser, Gaudenz

doi:10.1101/2020.11.30.404814

Cited by 14 publications

(14 citation statements)

References 44 publications

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“…Since the cell is morphologically eroded to a greater extent than it is dilated, the cell segmentation is effectively shrunk, reducing the effect of segmentation errors on later analysis. To detect dots of endocytosed collagen and dextran, we employed a previously published multiscale particle detector ( 82 ). We used scales of 1.5 to 4 pixels, an ⍰⍰= 0.01, and detected dots only inside the segmented cell.…”

Section: Methodsmentioning

confidence: 99%

Proteolysis-free cell migration through crowded environments via mechanical worrying

Welf

Driscoll

Sapoznik

et al. 2020

Preprint

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Migratory cells employ numerous strategies to navigate the very diverse 3D microenvironments found in vivo. These strategies are subdivided into those that create space by pericellular proteolysis of extracellular matrix (ECM) proteins and those that navigate existing spaces. We find that cells can employ an alternative mechanism by digging tunnels through 3D collagen networks without extracellular proteolysis. This is accomplished by persistent polarization of large dynamic membrane blebs at the closed end of the tunnel that repeatedly agitate the collagen, a process we termed mechanical worrying. We find that this agitation promotes breakage and internalization of collagen at the cell front along with extracellular fluid in a macropinocytosis-driven manner. Membrane blebs are short-lived relative to the timescale of migration, and thus their polarization is critical for persistent ablation of the ECM. We find that sustained interactions between the collagen at the cell front and small but persistent cortical adhesions induce PI-3 Kinase (PI3K) signaling that drives polarized bleb enlargement via the Rac1 – Arp2/3 pathway. This defines a mechanism for the reinforcement of bleb expansion against load, which enables precise ablation of mechanically unrestrained environments, such as those encountered in very compliant tissue.

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Section: Methodsmentioning

confidence: 99%

Proteolysis-free cell migration through crowded environments via mechanical worrying

Welf

Driscoll

Sapoznik

et al. 2020

Preprint

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“…Unlike in previous studies 31,54 the image intensity was not depth normalized prior to analysis, but was instead measured from the raw, undeconvolved image. To measure the localization of septin structures, rather than total septin, in figures 2C and 2E we employed a previously published multiscale stochastic filter to enhance dot-like structures 85 . With this filter, we used scales of 2 to 4 pixels, and an a = 0.01.…”

Section: D Cell Image Analysismentioning

confidence: 99%

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

Driscoll

et al. 2021

Preprint

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For most human cells, anchorage is a key necessity for survival. Cell-substrate adhesion activates diverse signaling pathways, without which cells undergo anoikis – a form of programmed cell death1. Acquisition of anoikis resistance is a pivotal step in cancer disease progression, as metastasizing cancer cells often lose firm attachment to surrounding tissue2–5. In these poorly attached states, cells often adopt rounded morphologies and form small hemispherical plasma membrane protrusions called blebs6–13. Bleb function has long been investigated in the context of amoeboid migration but is far less deeply examined in other scenarios14–19. Here we show by quantitative subcellular 3D imaging and manipulation of cell morphological states that blebbing triggers the formation of membrane-proximal signaling hubs that initiate signaling cascades leading to anoikis resistance. Specifically, in melanoma cells we discovered that blebbing generates plasma membrane contours that recruit curvature sensing septin proteins, which scaffold constitutively active mutant NRAS and effectors, driving the upregulation of ERK and PI3K signaling. Inhibition of blebs or septins has little effect on the survival of well-adhered cells, but in detached cells causes NRAS mislocalization, reduced MAPK and PI3K signaling, and ultimately, death. These data unveil an unanticipated morphological requirement for mutant NRAS to operate as an effective oncoprotein, suggesting novel clinical targets for the treatment of NRAS-driven melanoma. Furthermore, they define an unforeseen role for blebs as potent signaling organelles capable of integrating myriad cellular information flows into concerted signaling responses, in this case granting robust anoikis resistance.Abstract Figure

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“…Transfected cells were imaged every 12.5 sec over 5 min. Images were then processed using u-track software [20] and comets assessed for speed, lifetime and distance travelled.…”

Section: Analysis Of Cytoskeleton Dynamicsmentioning

confidence: 99%

A novel cytoskeletal action of xylosides

et al. 2022

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Proteoglycan glycosaminoglycan (GAG) chains are attached to a serine residue in the protein through a linkage series of sugars, the first of which is xylose. Xylosides are chemicals which compete with the xylose at the enzyme xylosyl transferase to prevent the attachment of GAG chains to proteins. These compounds have been employed at concentrations in the millimolar range as tools to study the role of GAG chains in proteoglycan function. In the course of our studies with xylosides, we conducted a dose-response curve for xyloside actions on neural cells. To our surprise, we found that concentrations of xylosides in the nanomolar to micromolar range had major effects on cell morphology of hippocampal neurons as well as of Neuro2a cells, affecting both actin and tubulin cytoskeletal dynamics. Such effects/morphological changes were not observed with higher xyloside concentrations. We found a dose-dependent alteration of GAG secretion by Neuro2a cells; however, concentrations of xylosides which were effective in altering neuronal morphology did not cause a large change in the rate of GAG chain secretion. In contrast, both low and high concentrations of xylosides altered HS and CS composition. RNAseq of treated cells demonstrated alterations in gene expression only after treatment with millimolar concentration of xylosides that had no effect on cell morphology. These observations support a novel action of xylosides on neuronal cells.

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u-track 3D: measuring and interrogating dense particle dynamics in three dimensions

Cited by 14 publications

References 44 publications

Proteolysis-free cell migration through crowded environments via mechanical worrying

Proteolysis-free cell migration through crowded environments via mechanical worrying

Blebs Promote Cell Survival by Assembling Oncogenic Signaling Hubs

A novel cytoskeletal action of xylosides

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