Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway

Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.

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Maturing autophagosomes are transported towards the cell periphery

10.21203/rs.3.rs-200706/v1 ◽

2021 ◽

Author(s):

Anna Hilverling ◽

Eva M. Szegö ◽

Elisabeth Dinter ◽

Diana Cozma ◽

Theodora Saridaki ◽

...

Keyword(s):

Subcellular Distribution ◽

Intracellular Trafficking ◽

Fluorescent Proteins ◽

Time Lapse ◽

Vesicle Transport ◽

Cell Periphery ◽

Time Lapse Microscopy ◽

Acidic Vesicles ◽

Luminal Ph ◽

Autophagosome Maturation

Abstract Autophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport.

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Maturing Autophagosomes are Transported Towards the Cell Periphery

Cellular and Molecular Neurobiology ◽

10.1007/s10571-021-01116-0 ◽

2021 ◽

Author(s):

Anna Hilverling ◽

Eva M. Szegö ◽

Elisabeth Dinter ◽

Diana Cozma ◽

Theodora Saridaki ◽

...

Keyword(s):

Intracellular Trafficking ◽

Fluorescent Proteins ◽

Time Lapse ◽

Cell Periphery ◽

Microtubule Organizing Center ◽

Time Lapse Microscopy ◽

Organizing Center ◽

Acidic Vesicles ◽

Luminal Ph ◽

Autophagosome Maturation

AbstractAutophagosome maturation comprises fusion with lysosomes and acidification. It is a critical step in the degradation of cytosolic protein aggregates that characterize many neurodegenerative diseases. In order to better understand this process, we studied intracellular trafficking of autophagosomes and aggregates of α-synuclein, which characterize Parkinson’s disease and other synucleinopathies. The autophagosomal marker LC3 and the aggregation prone A53T mutant of α-synuclein were tagged by fluorescent proteins and expressed in HEK293T cells and primary astrocytes. The subcellular distribution and movement of these vesicle populations were analyzed by (time-lapse) microscopy. Fusion with lysosomes was assayed using the lysosomal marker LAMP1; vesicles with neutral and acidic luminal pH were discriminated using the RFP-GFP “tandem-fluorescence” tag. With respect to vesicle pH, we observed that neutral autophagosomes, marked by LC3 or synuclein, were located more frequently in the cell center, and acidic autophagosomes were observed more frequently in the cell periphery. Acidic autophagosomes were transported towards the cell periphery more often, indicating that acidification occurs in the cell center before transport to the periphery. With respect to autolysosomal fusion, we found that lysosomes preferentially moved towards the cell center, whereas autolysosomes moved towards the cell periphery, suggesting a cycle where lysosomes are generated in the periphery and fuse to autophagosomes in the cell center. Unexpectedly, many acidic autophagosomes were negative for LAMP1, indicating that acidification does not require fusion to lysosomes. Moreover, we found both neutral and acidic vesicles positive for LAMP1, consistent with delayed acidification of the autolysosome lumen. Individual steps of aggregate clearance thus occur in dedicated cellular regions. During aggregate clearance, autophagosomes and autolysosomes form in the center and are transported towards the periphery during maturation. In this process, luminal pH could regulate the direction of vesicle transport. Graphic Abstract (1) Transport and location of autophagosomes depend on luminal pH: Acidic autophagosomes are preferentially transported to the cell periphery, causing more acidic autophagosomes in the cell periphery and more neutral autophagosomes at the microtubule organizing center (MTOC). (2) Autolysosomes are transported to the cell periphery and lysosomes to the MTOC, suggesting spatial segregation of lysosome reformation and autolysosome fusion. (3) Synuclein aggregates are preferentially located at the MTOC and synuclein-containing vesicles in the cell periphery, consistent with transport of aggregates to the MTOC for autophagy.

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Nudel functions in membrane traffic mainly through association with Lis1 and cytoplasmic dynein

The Journal of Cell Biology ◽

10.1083/jcb.200308058 ◽

2004 ◽

Vol 164 (4) ◽

pp. 557-566 ◽

Cited By ~ 83

Author(s):

Yun Liang ◽

Wei Yu ◽

Yan Li ◽

Zhenye Yang ◽

Xiumin Yan ◽

...

Keyword(s):

Heavy Chain ◽

Neuronal Migration ◽

Time Lapse ◽

Cytoplasmic Dynein ◽

Wild Type ◽

Dynein Heavy Chain ◽

Time Lapse Microscopy ◽

Dynein Motor ◽

Defective Mutant ◽

Directed Motility

Nudel and Lis1 appear to regulate cytoplasmic dynein in neuronal migration and mitosis through direct interactions. However, whether or not they regulate other functions of dynein remains elusive. Herein, overexpression of a Nudel mutant defective in association with either Lis1 or dynein heavy chain is shown to cause dispersions of membranous organelles whose trafficking depends on dynein. In contrast, the wild-type Nudel and the double mutant that binds to neither protein are much less effective. Time-lapse microscopy for lysosomes reveals significant reduction in both frequencies and velocities of their minus end–directed motions in cells expressing the dynein-binding defective mutant, whereas neither the durations of movement nor the plus end–directed motility is considerably altered. Moreover, silencing Nudel expression by RNA interference results in Golgi apparatus fragmentation and cell death. Together, it is concluded that Nudel is critical for dynein motor activity in membrane transport and possibly other cellular activities through interactions with both Lis1 and dynein heavy chain.

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Arabidopsis 4-COUMAROYL-COA LIGASE 8 contributes to the biosynthesis of the benzenoid ring of coenzyme Q in peroxisomes

Biochemical Journal ◽

10.1042/bcj20190688 ◽

2019 ◽

Vol 476 (22) ◽

pp. 3521-3532

Author(s):

Eric Soubeyrand ◽

Megan Kelly ◽

Shea A. Keene ◽

Ann C. Bernert ◽

Scott Latimer ◽

...

Keyword(s):

Oxidative Metabolism ◽

Time Course ◽

Reverse Genetics ◽

De Novo ◽

Coenzyme Q ◽

Control Level ◽

Wild Type ◽

Fluorescent Reporters ◽

Coa Ligase ◽

Peroxisomal Targeting

Plants have evolved the ability to derive the benzenoid moiety of the respiratory cofactor and antioxidant, ubiquinone (coenzyme Q), either from the β-oxidative metabolism of p-coumarate or from the peroxidative cleavage of kaempferol. Here, isotopic feeding assays, gene co-expression analysis and reverse genetics identified Arabidopsis 4-COUMARATE-COA LIGASE 8 (4-CL8; At5g38120) as a contributor to the β-oxidation of p-coumarate for ubiquinone biosynthesis. The enzyme is part of the same clade (V) of acyl-activating enzymes than At4g19010, a p-coumarate CoA ligase known to play a central role in the conversion of p-coumarate into 4-hydroxybenzoate. A 4-cl8 T-DNA knockout displayed a 20% decrease in ubiquinone content compared with wild-type plants, while 4-CL8 overexpression boosted ubiquinone content up to 150% of the control level. Similarly, the isotopic enrichment of ubiquinone's ring was decreased by 28% in the 4-cl8 knockout as compared with wild-type controls when Phe-[Ring-13C6] was fed to the plants. This metabolic blockage could be bypassed via the exogenous supply of 4-hydroxybenzoate, the product of p-coumarate β-oxidation. Arabidopsis 4-CL8 displays a canonical peroxisomal targeting sequence type 1, and confocal microscopy experiments using fused fluorescent reporters demonstrated that this enzyme is imported into peroxisomes. Time course feeding assays using Phe-[Ring-13C6] in a series of Arabidopsis single and double knockouts blocked in the β-oxidative metabolism of p-coumarate (4-cl8; at4g19010; at4g19010 × 4-cl8), flavonol biosynthesis (flavanone-3-hydroxylase), or both (at4g19010 × flavanone-3-hydroxylase) indicated that continuous high light treatments (500 µE m−2 s−1; 24 h) markedly stimulated the de novo biosynthesis of ubiquinone independently of kaempferol catabolism.

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Experience of using time-lapse microscopy in IVF and ICSI programs

Medical alphabet ◽

10.33667/2078-5631-2020-16-47-50 ◽

2020 ◽

pp. 47-50

Author(s):

N. V. Saraeva ◽

N. V. Spiridonova ◽

M. T. Tugushev ◽

O. V. Shurygina ◽

A. I. Sinitsyna

Keyword(s):

Pregnancy Rate ◽

Embryo Transfer ◽

Selection Procedure ◽

Time Lapse ◽

Single Embryo Transfer ◽

Main Group ◽

Clinical Pregnancy ◽

Control Group ◽

Time Lapse Microscopy

In order to increase the pregnancy rate in the assisted reproductive technology, the selection of one embryo with the highest implantation potential it is very important. Time-lapse microscopy (TLM) is a tool for selecting quality embryos for transfer. This study aimed to assess the benefits of single-embryo transfer of autologous oocytes performed on day 5 of embryo incubation in a TLM-equipped system in IVF and ICSI programs. Single-embryo transfer following incubation in a TLM-equipped incubator was performed in 282 patients, who formed the main group; the control group consisted of 461 patients undergoing single-embryo transfer following a traditional culture and embryo selection procedure. We assessed the quality of transferred embryos, the rates of clinical pregnancy and delivery. The groups did not differ in the ratio of IVF and ICSI cycles, average age, and infertility factor. The proportion of excellent quality embryos for transfer was 77.0% in the main group and 65.1% in the control group (p = 0.001). In the subgroup with receiving eight and less oocytes we noted the tendency of receiving more quality embryos in the main group (р = 0.052). In the subgroup of nine and more oocytes the quality of the transferred embryos did not differ between two groups. The clinical pregnancy rate was 60.2% in the main group and 52.9% in the control group (p = 0.057). The delivery rate was 45.0% in the main group and 39.9% in the control group (p > 0.050).

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Postnatal growth rate and gonadal development in circadian tau mutant hamsters reared in constant dim red light

Reproduction ◽

10.1530/reprod/118.2.327 ◽

2000 ◽

pp. 327-330 ◽

Cited By ~ 7

Author(s):

RJ Lucas ◽

JA Stirland ◽

YN Mohammad ◽

AS Loudon

Keyword(s):

Time Course ◽

Red Light ◽

Somatic Growth ◽

Postnatal Growth ◽

Gonadal Development ◽

Testicular Development ◽

Wild Type ◽

Similar Time ◽

Syrian Hamsters ◽

Body Weights

The role of the circadian clock in the reproductive development of Syrian hamsters (Mesocricetus auratus was examined in wild type and circadian tau mutant hamsters reared from birth to 26 weeks of age under constant dim red light. Testis diameter and body weights were determined at weekly intervals in male hamsters from 4 weeks of age. In both genotypes, testicular development, subsequent regression and recrudescence exhibited a similar time course. The age at which animals displayed reproductive photosensitivity, as exhibited by testicular regression, was unrelated to circadian genotype (mean +/- SEM: 54 +/- 3 days for wild type and 59 +/- 5 days for tau mutants). In contrast, our studies revealed a significant impact of the mutation on somatic growth, such that tau mutants weighed 18% less than wild types at the end of the experiment. Our study reveals that the juvenile onset of reproductive photoperiodism in Syrian hamsters is not timed by the circadian system.

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