The Journal of Neuroscience, June 10, 2015 • 35(23):8687–8688 • 8687 Journal Club Editor’s Note: These short, critical reviews of recent papers in the Journal, written exclusively by graduate students or postdoctoral fellows, are intended to summarize the important findings of the paper and provide additional insight and commentary. For more information on the format and purpose of the Journal Club, please see http://www.jneurosci.org/misc/ifa_features.shtml. Myosin II and Dynamin Control Actin Rings to Mediate Fission during Activity-Dependent Bulk Endocytosis Alexandros C. Kokotos and Darryl W. Low Centre for Integrative Physiology, University of Edinburgh, EH8 9XD, Scotland, United Kingdom Review of Gormal et al. Neuronal communication relies on tein syndapin I) and its GTPase activity that bulk endosomes surrounded by an neurotransmitter-filled synaptic vesicles facilitated bulk endosome formation and actin ring recovered their fluorescence (SVs) fusing with the presynaptic scission, respectively (Clayton et al., 2009). over time, indicating a connection to the plasma membrane during neuronal ac- However, these roles have recently been plasma membrane. Endosomes lacking tivity. After exocytosis, SVs must be re- questioned in experiments with dynamin I, such a ring failed to recover, indicating trieved locally for a continual supply of III double knock-out neurons (Wu et al., that fission was complete and they could SVs and neurotransmission to be main- 2014). Actin networks and PI-3 kinase, no longer internalize new dextran mole- tained. Clathrin-mediated endocytosis which phosphorylates phosphatidylinositol cules from the extracellular medium. (CME) is the dominant mode of SV re- 4,5-bisphosphate (PIP2), have also been Colocalization of LifeAct and glycosyl- trieval during low-intensity stimulation suggested to be essential for triggering phosphatidylinositol, a plasma membrane and is the best characterized form of en- ADBE (Holt et al., 2003). marker, provided further evidence that in- docytosis in many cell types and systems. Until recently, it was unknown whether vaginating bulk endosomes were still at- During high-intensity stimulation, a clathrin- ADBE was a neuron-specific pathway. In a tached to the plasma membrane. Further independent SV retrieval pathway named recent issue of The Journal of Neuroscience, studies on the nature of the actin rings activity-dependent bulk endocytosis (ADBE) Gormal and colleagues (2015) demon- showed that cortical actin stress filaments is also triggered (Clayton et al., 2008). The strated that ADBE is observed in bovine ad- partially depolymerized before ring forma- molecular mechanisms of ADBE are not renal chromaffin cells, and they provided tion and the rings formed exhibited a con- well defined. In ADBE, large invaginations insights about the molecular mechanisms of tractile nature. A probe that recognizes PIP form from the plasma membrane and 2 ADBE triggering. In this study, the authors indicated that an array of PIP2 microdo- quickly undergo scission to form bulk endo- used the fluid phase uptake of high- mains formed before the actin rings, sug- somes. Subsequently, new SVs are gener- molecular-weight fluorescent dextrans to gesting these microdomains may function ated from these endosomes. The large specifically track ADBE. This allows specific as hotspots for actin nucleation. Although GTPase dynamin I was initially proposed to labeling of endocytosis modes that internal- clustering of PIP , which has an established be central to ADBE triggering, because its 2 ize large amounts of plasma membrane, be- role in CME, was shown to precede actin activity-dependent dephosphorylation by cause the relatively large size of these ring formation, its potential mechanistic calcineurin (which allows the required in- polysaccharides makes internalization via role was not further investigated. teraction with the membrane-bending pro- large endosomes much more favorable The contractile ability of the actin rings compared with smaller vesicles. led the authors to probe the involvement Received March 26, 2015; revised April 24, 2015; accepted April 29, 2015. A.C.K. is supported by the Marie-Curie Initial Training Network “Nplast” Using this dextran assay, Gormal et al. of molecular motors in providing the con- award (project number 289581) and D.W.L. by a CIVIS Prize Studentship (2015) demonstrated that primary chro- traction force. Pharmacological inhibi- fromtheUniversityofEdinburgh.WethankProf.M.A.Cousinforhissuper- maffin cells internalized dextran after se- tion of myosin II blocked the formation of vision and stimulating discussion on the ADBE molecular mechanism and cretion was stimulated with nicotine or both bulk endosomes and actin rings. The physiology, and Dr. J.R. Marland for his helpful discussion and critical com- 2ϩ ments on the manuscript. Ba . The bulk endosomes formed had a few rings which did form did not exhibit The authors declare no competing financial interests. diameter larger than 1 ␮m and labeling the contractile ability needed to facilitate Correspondence should be addressed to Alexandros C. Kokotos, with LifeAct, an actin-binding peptide, fission. Similarly, the few bulk endosomes Centre for Integrative Physiology, Hugh Robson Building, 15 George showed that the endosomes were sur- formed were still connected to the plasma Square, University of Edinburgh, EH8 9XD, Scotland, U.K. E-mail: rounded by actin rings. Experiments us- membrane. The same phenotype was ob- [email protected]. DOI:10.1523/JNEUROSCI.1172-15.2015 ing dextrans and fluorescence recovery served when dynamin was inhibited using Copyright © 2015 the authors 0270-6474/15/358687-02$15.00/0 after photobleaching (FRAP) revealed either pharmacological or genetic appro- 8688 • J. Neurosci., June 10, 2015 • 35(23):8687–8688 Kokotos and Low • Journal Club aches. In summary, this study suggests a the endosomes differ in size by an order of to fully understand the molecular mechanism model of an acto-myosin II ring working in magnitude. Secondly, the timescale of endo- of ADBE. The study by Gormal et al. (2015) tandem to constrict and initiate the fission some fission is much slower, which allows highlights new avenues to explore. of bulk endosomes from the plasma mem- use of FRAP in this study. Furthermore, it is brane in ADBE. These rings are induced and unlikely that these endosomes generate new References contracted by myosin II and dynamin. granules after their fission from the plasma Boucrot E, Ferreira AP, Almeida-Souza L, Debard S, This proposed model for the molecu- membrane, since secretory granules are Vallis Y, Howard G, Bertot L, Sauvonnet N, Mc- lar mechanism of the constriction ring constantly generated from the trans-Golgi Mahon HT (2015) Endophilin marks and needs further clarification. Initial experi- network in chromaffin cells, whereas neuro- controls a clathrin-independent endocytic path- way. Nature 517:460–465. CrossRef Medline ments indicated that dextran-positive nal bulk endosomes produce new func- Chandrasekar I, Goeckeler ZM, Turney SG, Wang bulk endosomes appeared before the for- tional SVs locally. P, Wysolmerski RB, Adelstein RS, Bridgman mation of actin rings, which take part in Regardless of whether the endocytosis PC (2014) Nonmuscle myosin II is a critical fission. In later experiments, myosin II mode described by Gormal et al. (2015) is regulator of clathrin-mediated endocytosis. and dynamin inhibition prevented actin identical to neuronal ADBE, the findings Traffic 15:418–432. CrossRef Medline ring formation and dextran uptake. How shed new light on endocytic mechanisms. Clayton EL, Evans GJ, Cousin MA (2008) Bulk synaptic vesicle endocytosis is rapidly trig- then does ablation of the constricting ac- With new modes of endocytosis being re- gered during strong stimulation. J Neurosci tin ring result in no formation of bulk en- cently identified (Watanabe et al., 2013; 28:6627–6632. CrossRef Medline dosomes? One possibility is that dynamin Boucrot et al., 2015), it is becoming clear Clayton EL, Anggono V, Smillie KJ, Chau N, and myosin II also act in early steps of that some common mechanisms are con- Robinson PJ, Cousin MA (2009) The phospho- endocytosis to induce the invagination of served, especially with regards to scission dependent dynamin-syndapin interaction trig- bulk endosomes and then drive the actin of large invaginations from the plasma gers activity-dependent bulk endocytosis of ring formation to facilitate fission. In sup- membrane. An example is the role of dy- synaptic vesicles. J Neurosci 29:7706–7717. CrossRef Medline port, in CME either genetic ablation or namin. This study escapes the recent de- Gormal RS, Nguyen TH, Martin S, Papadopulos A, pharmacological inhibition of myosin II bate as to whether the GTPase activity of Meunier FA (2015) An acto-myosin II con- resulted in deficient clathrin-coated pit dynamin is required for scission during stricting ring initiates the fission of activity- (CCP) invagination (Chandrasekar et al., ADBE and provides an alternative view on dependent bulk endosomes in neurosecretory 2014). Similarly, dynamin is recruited to how dynamin may facilitate this process, cells. J Neurosci 35:1380–1389. CrossRef Medline nascent CCPs and overexpression of via an additional role in the formation and Gu C, Yaddanapudi S, Weins A, Osborn T, Reiser J, Pollak M, Hartwig J, Sever S (2010) Direct dominant-negative mutants as well as function of a constricting actin ring. The dynamin-actin interactions regulate the actin