Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

Kural, Cömert; Akatay, Ahmet Ata; Gaudin, Raphaël; Chen, Bi-Chang; Legant, Wesley R.; Betzig, Eric; Kirchhausen, Tom

doi:10.1091/mbc.e15-01-0055

Cited by 37 publications

(59 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, CHC depletion only partly inhibited the more rapid FA disassembly in Cul5-deficient cells (Figure 2—figure supplement 2a), suggesting that Cul5 inhibits a distinct mechanism of disassembly that is clathrin-independent. Consistent with this interpretation, clathrin-coated pits were missing from the leading lamellipodium of migrating Cul5-deficient cells (Figure 2—figure supplement 2b), as recently reported for other cell types (Kural et al, 2015). This suggests that Cul5 regulates FA disassembly through mechanisms unrelated to clathrin-mediated endocytosis.…”

Section: Resultssupporting

confidence: 89%

“…However, our evidence suggests that MT-dependent integrin endocytosis is not involved in the SFK-Cas FA disassembly pathway. Clathrin-coated pits are absent from the ventral surface of the leading edge of motile glioblastoma cells (Kural et al, 2015) and were absent from the long leading lamellipodium of Cul5-deficient MCF10A cells. Moreover, clathrin knockdown inhibited the slow FA disassembly in normal HeLa cells but not the rapid FA disassembly in Cul5-deficient HeLa cells.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

Teckchandani

Cooper

2016

eLife

View full text Add to dashboard Cite

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. Here we show that suppressor of cytokine signaling 6 (SOCS6) and Cullin 5, two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or if cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration.DOI: http://dx.doi.org/10.7554/eLife.17440.001

show abstract

Section: Resultssupporting

confidence: 89%

Section: Discussionmentioning

confidence: 99%

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

Teckchandani

Cooper

2016

eLife

View full text Add to dashboard Cite

show abstract

“…This cell line represents a physiologically relevant model to study JUNV biology because JUNV-induced Argentine hemorrhagic fever manifests with neurological symptoms (1). While they are less relevant, breast cancer SUM159 cells were used here because we have previously optimized gene-editing strategies to express fluorescently labeled endogenous proteins in these cells (27,33). Here we showed that we can also manipulate the genome of SVG-A cells by inserting a fluorescent EGFP tag into the N terminus of the endosomal small GTPases Rab5c and Rab7a (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…To this end, we had to identify new human cellular models compatible with such techniques because the genome of Vero cells (derived from the green African monkey) has not been fully sequenced and at present is not suited for use in efficient gene-editing approaches. We had successful experiences in editing the genes of cells of the human breast cancer cell line SUM159 (27,32,33) and therefore evaluated SUM159 cells for permissiveness and compared them to the Vero and A549 cells routinely used in JUNV infection assays. In addition, we also tested SVG-A human astroglial cells as a potential host relevant for JUNV physiopathological studies.…”

Section: Characterization Of New Cell Line Models Suitable For Junv Imentioning

confidence: 99%

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

Chou

Cuevas

Carocci

et al. 2016

J Virol

Self Cite

View full text Add to dashboard Cite

Virus entry into cells is a multistep process that often requires the subversion of subcellular machineries. A more complete understanding of these steps is necessary to develop new antiviral strategies. While studying the potential role of the actin network and one of its master regulators, the small GTPase Cdc42, during Junin virus (JUNV) entry, we serendipitously uncovered the small molecule ZCL278, reported to inhibit Cdc42 function as an entry inhibitor for JUNV and for vesicular stomatitis virus, lymphocytic choriomeningitis virus, and dengue virus but not for the nonenveloped poliovirus. Although ZCL278 did not interfere with JUNV attachment to the cell surface or virus particle internalization into host cells, it prevented the release of JUNV ribonucleoprotein cores into the cytosol and decreased pH-mediated viral fusion with host membranes. We also identified SVG-A astroglial cell-derived cells to be highly permissive for JUNV infection and generated new cell lines expressing fluorescently tagged Rab5c or Rab7a or lacking Cdc42 using clustered regularly interspaced short palindromic repeat (CRISPR)-caspase 9 (Cas9) gene-editing strategies. Aided by these tools, we uncovered that perturbations in the actin cytoskeleton or Cdc42 activity minimally affect JUNV entry, suggesting that the inhibitory effect of ZCL278 is not mediated by ZCL278 interfering with the activity of Cdc42. Instead, ZCL278 appears to redistribute viral particles from endosomal to lysosomal compartments. ZCL278 also inhibited JUNV replication in a mouse model, and no toxicity was detected. Together, our data suggest the unexpected antiviral activity of ZCL278 and highlight its potential for use in the development of valuable new tools to study the intracellular trafficking of pathogens. IMPORTANCEThe Junin virus is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are at risk. Limited options are currently available to treat infections by Junin virus or other viruses of the Arenaviridae, making the identification of additional tools, including small-molecule inhibitors, of great importance. How Junin virus enters cells is not yet fully understood. Here we describe new cell culture models in which the cells are susceptible to Junin virus infection and to which we applied CRISPR-Cas9 genome engineering strategies to help characterize early steps during virus entry. We also uncovered ZCL278 to be a new antiviral small molecule that potently inhibits the cellular entry of the Junin virus and other enveloped viruses. Moreover, we show that ZCL278 also functions in vivo, thereby preventing Junin virus replication in a mouse model, opening the possibility for the discovery of ZCL278 derivatives of therapeutic potential. T he Junin virus (JUNV) is an important member of the NewWorld arenavirus family (clade B) and is responsible for outbreaks of Argentine hemorrhagic fever in South America, where 5 million people are considered to be at risk (1). The reservoir of JUNV is the rodent Calomy...

show abstract

“…1). We have worked with over fifty different groups to apply these tools in areas including: mitotic spindle alignment during asymmetric stem cell division [2]; actomyosin contractions driving the initial gastrulation of C. elegans embryos [3]; binding kinetics of single transcription factor molecules to DNA in live stem cells [4]; dynamic, heterogeneous remodeling of P granule proteins in C. elegans embryos [5]; asymmetric formation of clathrin-coated pits on the dorsal /ventral surfaces at the leading edge of motile cells [6]; rapid 3D redistribution of actin in T cells during the formation for the immunological synapse [7]; and spatiotemporal quantification of microtubule growth tracks throughout the cellular volume at all mitotic stages [8].…”

mentioning

confidence: 99%

Imaging Cellular Structure and Dynamics from Molecules to Organisms

Betzig

2017

Microsc Microanal

Self Cite

View full text Add to dashboard Cite

The hallmark of life is that it is animate. Every living thing is a complex pocket of reduced entropy through which matter and energy flow continuously. Thus, although structural imaging is informative, a more complete understanding of the molecular basis of cellular physiology requires high-resolution imaging of the dynamics of the cell in its native state across all four dimensions of spacetime simultaneously.Unfortunately, several factors conspire to render such unperturbed, physiological 4D imaging difficult. First, as powerful as genetically encoded fluorescent proteins have become, until recently they have rarely been used at endogenous expression levels, and therefore can upset the homeostatic balance of the cell. New genome editing technologies, specifically CRISPR / CAS9, address this problem. Second, conventional live cell imaging tools such as spinning disk confocal microscopy are too slow to study fast cellular processes across cellular volumes, create out-of-focus photo-induced damage and fluorescence photobleaching, and subject the cell at the point of measurement (i.e., the excitation focus) to peak intensities orders of magnitude beyond that under which life evolved. In the past few years, we have used "non-diffracting" beams, specifically Bessel beams and 2D optical lattices, to create ultra-thin light sheets capable of imaging of sub-cellular dynamics in 3D across whole cells and small embryos with near-isotropic resolution at up to 1000 image planes/sec over hundreds of time points ([1], Fig. 1). We have worked with over fifty different groups to apply these tools in areas including: mitotic spindle alignment during asymmetric stem cell division Finally, much of the contribution of optical microscopy to cell biology has come from observing individual cells cultured onto glass substrates, and yet it is certain that they did not evolve there. True physiological imaging likely requires studying cells in their parent organisms, where all the external environmental cues that drive gene expression, and hence their structural and functional phenotypes, are present. However, such imaging is compromised by the highly inhomogeneous refractive index of most biological tissues, which distorts light rays and thereby degrades both resolution and signal. We have adopted methods of adaptive optics (AO), initially developed in astronomy, to recover diffractionlimited performance deep within living systems ([9], Fig. 2, left and bottom), and have recently combined AO on both the excitation and detection arms of our lattice light sheet microscope to image sub-cellular dynamics noninvasively within multicellular systems such as developing zebrafish embryos (unpublished, Fig. 2, upper right).

show abstract

Asymmetric formation of coated pits on dorsal and ventral surfaces at the leading edges of motile cells and on protrusions of immobile cells

Cited by 37 publications

References 55 publications

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

Identification and Characterization of a Novel Broad-Spectrum Virus Entry Inhibitor

Imaging Cellular Structure and Dynamics from Molecules to Organisms

Contact Info

Product

Resources

About