Gaining independence
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RESEARCH HIGHLIGHTS NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MARCH 2005 | 191 AD test strikes gold A nanoparticle test reported in the Proceedings of the National Academy of Sciences could herald the advent of a living diagnosis for Alzheimer’s disease (AD). The test detects the prevalence of amyloid-β- derived diffusible ligands (ADDLs), and is a million times more sensitive than other approaches. ADDLs are “…invisible to conventional neuropathology, but their presence or absence may be the real determinants of memory loss,” according to William L. Klein, a member of the research team (Science News Online, 5 February 2005), and they have been shown to target synapses well before symptoms can be spotted at present. At the moment, a firm diagnosis of AD requires an autopsy, and other forms of diagnosis, such as memory tests and brain scans, fall short of 100% accuracy. Although ADDLs were suspected to be present in the cerebrospinal fluid, no tests were sensitive enough to detect their presence there. The approach taken by Dimitra Georganopoulou and colleagues used antibodies directed against ADDL. Some of the antibodies were attached to iron particles, and could therefore be extracted using a magnetic field. Other antibodies, which were attached to gold nanoparticles, could also hitch a lift in the extraction process. Along with the gold were ‘barcode’ DNA strands, which, when released by dehybridization, bound to complementary DNA on a glass plate and were detected using a highly sensitive scanometric method. Faced with the challenge of trying out the technique on easier-to-obtain blood and urine samples, in which ADDL concentrations are expected to be far lower, the team remains confident. “We’re nowhere near the limits of the test’s sensitivity.” (NewScientist.com, 31 January 2005.) Katrin Bussell WEB WATCH The vast majority of our present knowledge about endocytic pathways concerns classic clathrin-dependent endocytosis. Clathrin-independent pathways are poorly understood by comparison, although, in recent years, evidence has accumulated for clathrin-independent pathways that are mediated by caveolae (caveolin-1- containing structures) or lipid rafts. Now, in The Journal of Cell Biology, two papers — the first by Parton and colleagues, the second by Helenius and co-workers — provide new insights into endocytic pathways that are inde- pendent of both clathrin and caveolae. In the first paper, Parton and col- leagues studied the internalization of the cholera-toxin binding subunit (CTB). It is known that CTB can be endocytosed by clathrin-independent mechanisms, but whether caveolae are involved has remained controver- sial. The authors first developed an ultrastructural assay that allowed them to identify budded caveolin-1- positive structures for the first time. In wild-type mouse embryonic fibro- blasts (MEFs), they found that a small fraction of CTB- and caveolin-1- positive structures budded from the plasma membrane, and that this budding could be stimulated. However, they also showed that this is not the predominant pathway for CTB uptake in MEFs, as CTB trafficking to the Golgi was found to be identical in wild-type and caveolin- 1 –/– MEFs. In addition, they showed that blocking clathrin-mediated endo- cytosis in caveolin-1 –/– MEFs only partially inhibited the Golgi accumu- lation of CTB. It therefore seems that a significant proportion of CTB up- take can occur through a pathway that is independent of caveolae and clathrin, and Parton and colleagues speculate that this might represent a primordial endocytic route. Through further characterization of this pathway, they found that the internalization was dynamin and Arf6 independent, but relatively cholesterol dependent. They also showed that the endocytic vehicles were uncoated tubular/ring-like struc- tures with diameters of 40–80 nm. These structures were found to con- tain CTB, glycosylphosphatidylino- sitol-anchored proteins (GPI-APs) and fluid-phase markers. They are therefore probably GEECs (GPI-AP- enriched early endosomal compart- ments), structures that have been previously shown to be involved in the CDC42-dependent uptake of GPI-APs and fluid-phase markers. In the second paper, Helenius and co-workers studied the internalization of simian virus-40 (SV40), which is known to be able to enter cells through a clathrin-independent, caveolae- dependent pathway. However, by using a caveolin-1-deficient cell line and caveolin-1 –/– MEFs and by block- ing clathrin-mediated endocytosis, they found that SV40 can also enter cells through a clathrin- and caveolae- independent pathway. This pathway was more rapid than caveolae-depen- dent uptake and, by using various inhibitors, they showed that it was dynamin-2 and Arf6 independent, but cholesterol and tyrosine-kinase dependent. The authors further characterized this pathway and found that, after binding to the plasma membrane, SV40 rapidly associated with lipid rafts and then remained associated with these rafts. They also showed that SV40 was first internalized into small, tight-fitting vesicles (60 nm in diameter) before being transported to pH-neutral organelles that resembled caveosomes but were devoid of cave- olin. From these caveosome-like struc- tures, which they speculate might be where the caveolae-dependent and caveolae-independent pathways inter- sect, SV40 then moved through micro- tubule-mediated vesicular transport to the endoplasmic reticulum (ER) — a step that was required for infectivity. Helenius and co-workers have therefore identified an endocytic path- way that can transport SV40 from the plasma membrane to the ER in a clathrin- and caveolae-independent manner. And, although these two papers have provided new informa- tion regarding clathrin- and caveolae- independent endocytic pathways, they also raise the question, just how many clathrin-independent pathways are there? Rachel Smallridge References and links ORIGINAL RESEARCH PAPERS Kirkham, M. et al. Ultrastructural identification of uncoated caveolin-independent early endocytic vehicles. J. Cell Biol. 168, 465–476 (2005) | Damm, E.-M. et al. Clathrin- and caveolin-1- independent endocytosis: entry of simian virus 40 into cells devoid of caveolae. J. Cell Biol. 168, 477–488 (2005) FURTHER READING Parton, R. G. & Richards, A. A. Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms. Traffic 4, 724–738 (2003) WEB SITES Ari Helenius’ laboratory: http://www.bc.biol.ethz.ch/people/groups/arih/ Robert Parton’s laboratory: http://www.imb.uq.edu.au/index.html?page=116 88&pid=12015 Gaining independence ENDOCYTOSIS
Transcript of Gaining independence
R E S E A R C H H I G H L I G H T S
NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 6 | MARCH 2005 | 191
AD test strikes goldA nanoparticle test reportedin the Proceedings of theNational Academy of Sciencescould herald the advent of aliving diagnosis for Alzheimer’sdisease (AD). The test detectsthe prevalence of amyloid-β-derived diffusible ligands(ADDLs), and is a million timesmore sensitive than otherapproaches.
ADDLs are “…invisible toconventional neuropathology,but their presence orabsence may be the realdeterminants of memoryloss,” according to William L.Klein, a member of theresearch team (ScienceNews Online, 5 February2005), and they have beenshown to target synapseswell before symptoms can bespotted at present. At themoment, a firm diagnosis ofAD requires an autopsy, andother forms of diagnosis,such as memory tests andbrain scans, fall short of100% accuracy.
Although ADDLs weresuspected to be present inthe cerebrospinal fluid, notests were sensitive enough todetect their presence there.The approach taken byDimitra Georganopoulou andcolleagues used antibodiesdirected against ADDL.Some of the antibodies wereattached to iron particles,and could therefore beextracted using a magneticfield. Other antibodies,which were attached to goldnanoparticles, could alsohitch a lift in the extractionprocess. Along with the goldwere ‘barcode’ DNA strands,which, when released bydehybridization, bound tocomplementary DNA on aglass plate and were detectedusing a highly sensitivescanometric method.
Faced with the challenge oftrying out the technique oneasier-to-obtain blood andurine samples, in whichADDL concentrations areexpected to be far lower, the team remains confident.“We’re nowhere near thelimits of the test’s sensitivity.”(NewScientist.com, 31 January 2005.)
Katrin Bussell
WEB WATCH
The vast majority of our presentknowledge about endocytic pathwaysconcerns classic clathrin-dependentendocytosis. Clathrin-independentpathways are poorly understood by comparison, although, in recentyears, evidence has accumulated forclathrin-independent pathways thatare mediated by caveolae (caveolin-1-containing structures) or lipid rafts.Now, in The Journal of Cell Biology,two papers — the first by Parton andcolleagues, the second by Helenius andco-workers — provide new insightsinto endocytic pathways that are inde-pendent of both clathrin and caveolae.
In the first paper, Parton and col-leagues studied the internalization ofthe cholera-toxin binding subunit(CTB). It is known that CTB can beendocytosed by clathrin-independentmechanisms, but whether caveolaeare involved has remained controver-sial. The authors first developed anultrastructural assay that allowedthem to identify budded caveolin-1-positive structures for the first time.In wild-type mouse embryonic fibro-blasts (MEFs), they found that asmall fraction of CTB- and caveolin-1-positive structures budded from theplasma membrane, and that thisbudding could be stimulated.
However, they also showed thatthis is not the predominant pathwayfor CTB uptake in MEFs, as CTBtrafficking to the Golgi was found tobe identical in wild-type and caveolin-1–/– MEFs. In addition, they showedthat blocking clathrin-mediated endo-cytosis in caveolin-1–/– MEFs onlypartially inhibited the Golgi accumu-lation of CTB. It therefore seems thata significant proportion of CTB up-take can occur through a pathwaythat is independent of caveolae andclathrin, and Parton and colleaguesspeculate that this might represent aprimordial endocytic route.
Through further characterizationof this pathway, they found that theinternalization was dynamin andArf6 independent, but relativelycholesterol dependent. They alsoshowed that the endocytic vehicles
were uncoated tubular/ring-like struc-tures with diameters of 40–80 nm.These structures were found to con-tain CTB, glycosylphosphatidylino-sitol-anchored proteins (GPI-APs)and fluid-phase markers. They aretherefore probably GEECs (GPI-AP-enriched early endosomal compart-ments), structures that have beenpreviously shown to be involved inthe CDC42-dependent uptake ofGPI-APs and fluid-phase markers.
In the second paper, Helenius andco-workers studied the internalizationof simian virus-40 (SV40), which isknown to be able to enter cells througha clathrin-independent, caveolae-dependent pathway. However, byusing a caveolin-1-deficient cell lineand caveolin-1–/– MEFs and by block-ing clathrin-mediated endocytosis,they found that SV40 can also entercells through a clathrin- and caveolae-independent pathway. This pathwaywas more rapid than caveolae-depen-dent uptake and, by using variousinhibitors, they showed that it wasdynamin-2 and Arf6 independent,but cholesterol and tyrosine-kinasedependent.
The authors further characterizedthis pathway and found that, afterbinding to the plasma membrane,SV40 rapidly associated with lipidrafts and then remained associated
with these rafts. They also showedthat SV40 was first internalized intosmall, tight-fitting vesicles (60 nm indiameter) before being transported topH-neutral organelles that resembledcaveosomes but were devoid of cave-olin. From these caveosome-like struc-tures, which they speculate might bewhere the caveolae-dependent andcaveolae-independent pathways inter-sect, SV40 then moved through micro-tubule-mediated vesicular transport tothe endoplasmic reticulum (ER) — astep that was required for infectivity.
Helenius and co-workers havetherefore identified an endocytic path-way that can transport SV40 from theplasma membrane to the ER in aclathrin- and caveolae-independentmanner. And, although these twopapers have provided new informa-tion regarding clathrin- and caveolae-independent endocytic pathways, theyalso raise the question, just how manyclathrin-independent pathways arethere?
Rachel SmallridgeReferences and links
ORIGINAL RESEARCH PAPERS Kirkham, M. et al. Ultrastructural identification ofuncoated caveolin-independent early endocyticvehicles. J. Cell Biol. 168, 465–476 (2005) |Damm, E.-M. et al. Clathrin- and caveolin-1-independent endocytosis: entry of simian virus 40into cells devoid of caveolae. J. Cell Biol. 168,477–488 (2005)FURTHER READING Parton, R. G. & Richards, A. A. Lipid rafts and caveolae as portalsfor endocytosis: new insights and commonmechanisms. Traffic 4, 724–738 (2003)WEB SITESAri Helenius’ laboratory:http://www.bc.biol.ethz.ch/people/groups/arih/Robert Parton’s laboratory:http://www.imb.uq.edu.au/index.html?page=11688&pid=12015
Gaining independence
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