Non-specificity of Pitstop 2 in clathrin-mediated endocytosis

Willox, Anna K.; Sahraoui, Yasmina M. E.; Royle, Stephen

doi:10.1242/bio.20147955

Cited by 77 publications

(65 citation statements)

References 25 publications

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“…This finding was further verified by using the small-molecule inhibitor of clathrin, Pitstop 2 (von Kleist et al, 2011). Pitstop 2 inhibits binding of endocytic accessory proteins (such as amphiphysin) to the Nterminal domain of clathrin, thereby perturbing proper assembly and internalization of the clathrin network (von Kleist et al, 2011), although Pitstop 2 has also been reported to inhibit clathrinindependent endocytosis (Dutta et al, 2012;Willox et al, 2014) (see more in Discussion). Cells were treated with 20 µM Pitstop 2, which did not impact cell viability and effectively inhibited EGF internalization ( Fig.…”

Section: Resultsmentioning

confidence: 77%

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

Rosselli‐Murai

Yates

Yoshida

et al. 2018

Journal of Cell Science

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Defective endocytosis and vesicular trafficking of signaling receptors has recently emerged as a multifaceted hallmark of malignant cells. Clathrin-coated pits (CCPs) display highly heterogeneous dynamics on the plasma membrane where they can take from 20 s to over 1 min to form cytosolic coated vesicles. Despite the large number of cargo molecules that traffic through CCPs, it is not well understood whether signaling receptors activated in cancer, such as epidermal growth factor receptor (EGFR), are regulated through a specific subset of CCPs. The signaling lipid phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P], which is dephosphorylated by phosphatase and tensin homolog (PTEN), is a potent tumorigenic signaling lipid. By using total internal reflection fluorescence microscopy and automated tracking and detection of CCPs, we found that EGF-bound EGFR and PTEN are enriched in a distinct subset of short-lived CCPs that correspond with clathrin-dependent EGF-induced signaling. We demonstrated that PTEN plays a role in the regulation of CCP dynamics. Furthermore, increased PI(3,4,5)P resulted in higher proportion of short-lived CCPs, an effect that recapitulates PTEN deletion. Altogether, our findings provide evidence for the existence of short-lived 'signaling-capable' CCPs.

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Section: Resultsmentioning

confidence: 77%

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

Rosselli‐Murai

Yates

Yoshida

et al. 2018

Journal of Cell Science

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“…First, whole E8.0 cord stage embryos were cultured (WEC) for 3 hours and treated with Pitstop 2, a drug that inhibits both clathrin-mediated and clathrin-independent endocytosis 16, 17 . We found that adhesions were cleared from the apical membrane in both control and Pitstop 2-treated embryos (Online Figure II A–E).…”

Section: Resultsmentioning

confidence: 99%

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II

Barry

Koo

Norden

et al. 2016

Circulation Research

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Rationale Vascular tubulogenesis is essential to cardiovascular development. Within initial vascular cords of endothelial cells (ECs), apical membranes are established and become cleared of cell-cell junctions, thereby allowing continuous central lumens to open. Rasip1 is required for apical junction clearance, as well as for regulation of Rho GTPase activity. However, it remains unknown how activities of different Rho GTPases are coordinated by Rasip1 to direct tubulogenesis. Objective The aim of this study is to determine the mechanisms downstream of Rasip1 that drive vascular tubulogenesis. Methods and Results Using conditional mouse mutant models and pharmacological approaches, we dissect GTPase pathways downstream of Rasip1. We show that clearance of EC apical junctions during vascular tubulogenesis depends on Ras interacting protein 1 (Rasip1), as well as the GTPase Cdc42 and the kinase Pak4. Genetic deletion of Rasip1 or Cdc42, or inhibition of Pak4, all block EC tubulogenesis. By contrast, inactivation of RhoA signaling leads to vessel overexpansion, implicating actomyosin contractility in control of lumen diameter. Interestingly, blocking activity of NMII either prior to, or after, lumen morphogenesis results in dramatically different tubulogenesis phenotypes, suggesting time-dependent roles. Conclusions Rasip1 controls different pools of GTPases, which in turn regulate different pools of NMII to coordinate junction clearance (remodeling) and actomyosin contractility during vascular tubulogenesis. Rasip1 promotes activity of Cdc42 to activate Pak4, which in turn activates NMII, clearing apical junctions. Once lumens open, Rasip1 suppresses actomyosin contractility via inhibition of RhoA by Arhgap29, allowing controlled expansion of vessel lumens during embryonic growth. These findings elucidate the stepwise processes regulated by Rasip1 through downstream Rho GTPases and NMII.

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“…Our mechanistic conclusions are based on pharmacological inhibitors and are thus limited by potential off-target effects (Park et al, 2013; Willox et al, 2014). We also note that we tested only a single pharmacological inhibitor per endocytosis protein.…”

Section: Discussionmentioning

confidence: 99%

Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses

Delvendahl

Vyleta

Gersdorff

et al. 2016

Neuron

125

155

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The fusion of neurotransmitter-filled vesicles during synaptic transmission is balanced by endocytotic membrane retrieval. Despite extensive research, the speed and mechanisms of synaptic vesicle endocytosis have remained controversial. Here, we establish low-noise time-resolved membrane capacitance measurements that allow monitoring changes in surface membrane area elicited by single action potentials and stronger stimuli with high-temporal resolution at physiological temperature in individual bonafide mature central synapses. We show that single action potentials trigger very rapid endocytosis, retrieving presynaptic membrane with a time constant of 470 ms. This fast endocytosis is independent of clathrin, but mediated by dynamin and actin. In contrast, stronger stimuli evoke a slower mode of endocytosis that is clathrin-, dynamin-, and actin-dependent. Furthermore, the speed of endocytosis is highly temperature-dependent with a Q10 of ~3.5. These results demonstrate that distinct molecular modes of endocytosis with markedly different kinetics operate at central synapses.

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Non-specificity of Pitstop 2 in clathrin-mediated endocytosis

Cited by 77 publications

References 25 publications

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

Loss of PTEN promotes formation of signaling-capable clathrin-coated pits

Rasip1-Mediated Rho GTPase Signaling Regulates Blood Vessel Tubulogenesis via Nonmuscle Myosin II

Fast, Temperature-Sensitive and Clathrin-Independent Endocytosis at Central Synapses

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