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Multibudded tubules formed by COPII on artificial liposomes.

ABSTRACT: COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to low curvatures. The COPII proteins induced beads-on-a-string-like constricted tubules, similar to those previously observed in cells. We speculate about a mechanical pathway for vesicle fission from these multibudded COPII-coated tubules, considering the possibility that withdrawal of the Sar1 amphipathic helix upon GTP hydrolysis leads to lipid bilayer destabilization resulting in fission.

SUBMITTER: Bacia K

PROVIDER: S-EPMC3216505 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Multibudded tubules formed by COPII on artificial liposomes.

Bacia Kirsten K Futai Eugene E Prinz Simone S Meister Annette A Daum Sebastian S Glatte Daniela D Briggs John A G JA Schekman Randy R

Scientific reports 20110617

COPII-coated vesicles form at the endoplasmic reticulum for cargo transport to the Golgi apparatus. We used in vitro reconstitution to examine the roles of the COPII scaffold in remodeling the shape of a lipid bilayer. Giant Unilamellar Vesicles were examined using fast confocal fluorescence and cryo-electron microscopy in order to avoid separation steps and minimize mechanical manipulation. COPII showed a preference for high curvature structures, but also sufficient flexibility for binding to l ...[more]

PMID: 22355536

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Project description:Study questionCan transplanted primate testicular cells form seminiferous tubules de novo, supporting complete spermatogenesis?Summary answerCryopreserved testicular cells from a prepubertal monkey can reorganize in an adult monkey recipient testis forming de novo seminiferous tubular cords supporting complete spermatogenesis.What is known alreadyDe novo morphogenesis of testicular tissue using aggregated cells from non-primate species grafted either subcutaneously or in the testis can support spermatogenesis.Study design, size, durationTwo postpubertal rhesus monkeys (Macaca mulatta) were given testicular irradiation. One monkey was given GnRH-antagonist treatment from 8 to 16 weeks after irradiation, while the other received sham injections. At 16 weeks, cryopreserved testicular cells from two different prepubertal monkeys [43 × 106 viable (Trypan-blue excluding) cells in 260 μl, and 80 × 106 viable cells in 400 μl] were transplanted via ultrasound-guided injections to one of the rete testis in each recipient, and immune suppression was given. The contralateral testis was sham transplanted. Testes were analyzed 9 months after transplantation.Participants/materials, setting, methodsSpermatogenic recovery was assessed by testicular volume, weight, histology and immunofluorescence. Microsatellite genotyping of regions of testicular sections obtained by LCM determined whether the cells were derived from the host or transplanted cells.Main results and the role of chanceTransplanted testis of the GnRH-antagonist-treated recipient, but not the sham-treated recipient, contained numerous irregularly shaped seminiferous tubular cords, 89% of which had differentiating germ cells, including sperm in a few of them. The percentages of donor genotype in different regions of this testis were as follows: normal tubule, 0%; inflammatory, 0%; abnormal tubule region, 67%; whole interior of abnormal tubules, >99%; adluminal region of the abnormal tubules, 92%. Thus, these abnormal tubules, including the enclosed germ cells, were derived de novo from the donor testicular cells.Large scale dataNot applicable.Limitations, reasons for cautionThe de novo tubules were observed in only one out of the two monkeys transplanted with prepubertal donor testicular cells.Wider implications of the findingsThese findings may represent a promising strategy for restoration of fertility in male childhood cancer survivors. The approach could be particularly useful in those exposed to therapeutic agents that are detrimental to the normal development of the tubule somatic cells affecting the ability of the endogenous tubules to support spermatogenesis, even from transplanted spermatogonial stem cells.Study funding/competing interest(s)This work was supported by research grants P01 HD075795 from Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD/NIH) to K.E.O and Cancer Center Support Grant P30 CA016672 from NCI/NIH to The University of Texas MD Anderson Cancer Center. The authors declare that they have no competing interests.

Project description:We study the experimentally measured ciprofloxacin antibiotic diffusion through a gel-like artificial sputum medium (ASM) mimicking physiological conditions typical for a cystic fibrosis layer, in which regions occupied by Pseudomonas aeruginosa bacteria are present. To quantify the antibiotic diffusion dynamics we employ a phenomenological model using a subdiffusion-absorption equation with a fractional time derivative. This effective equation describes molecular diffusion in a medium structured akin Thompson's plumpudding model; here the 'pudding' background represents the ASM and the 'plums' represent the bacterial biofilm. The pudding is a subdiffusion barrier for antibiotic molecules that can affect bacteria found in plums. For the experimental study we use an interferometric method to determine the time evolution of the amount of antibiotic that has diffused through the biofilm. The theoretical model shows that this function is qualitatively different depending on whether or not absorption of the antibiotic in the biofilm occurs. We show that the process can be divided into three successive stages: (1) only antibiotic subdiffusion with constant biofilm parameters, (2) subdiffusion and absorption of antibiotic molecules with variable biofilm transport parameters, (3) subdiffusion and absorption in the medium but the biofilm parameters are constant again. Stage 2 is interpreted as the appearance of an intensive defence build-up of bacteria against the action of the antibiotic, and in the stage 3 it is likely that the bacteria have been inactivated. Times at which stages change are determined from the experimentally obtained temporal evolution of the amount of antibiotic that has diffused through the ASM with bacteria. Our analysis shows good agreement between experimental and theoretical results and is consistent with the biologically expected biofilm response. We show that an experimental method to study the temporal evolution of the amount of a substance that has diffused through a biofilm is useful in studying the processes occurring in a biofilm. We also show that the complicated biological process of antibiotic diffusion in a biofilm can be described by a fractional subdiffusion-absorption equation with subdiffusion and absorption parameters that change over time.

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Multibudded tubules formed by COPII on artificial liposomes.

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Multibudded tubules formed by COPII on artificial liposomes.

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