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| 2. |
- Au, Catherine E, et al.
(författare)
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Organellar proteomics to create the cell map.
- 2007
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Ingår i: Current opinion in cell biology. - : Elsevier BV. - 0955-0674. ; 19:4, s. 376-85
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Tidskriftsartikel (refereegranskat)abstract
- The elucidation of a complete, accurate, and permanent representation of the proteome of the mammalian cell may be achievable piecemeal by an organellar based approach. The small volume of organelles assures high protein concentrations. Providing isolated organelles are homogenous, this assures reliable protein characterization within the sensitivity and dynamic range limits of current mass spec based analysis. The stochastic aspect of peptide selection by tandem mass spectrometry for sequence determination by fragmentation is dealt with by multiple biological replicates as well as by prior protein separation on 1-D gels. Applications of this methodology to isolated synaptic vesicles, clathrin coated vesicles, endosomes, phagosomes, endoplasmic reticulum, and Golgi apparatus, as well as Golgi-derived COPI vesicles, have led to mechanistic insight into the identity and function of these organelles.
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| 3. |
- Bell, Alexander W, et al.
(författare)
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The protein microscope: incorporating mass spectrometry into cell biology.
- 2007
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Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 4:10, s. 783-4
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Tidskriftsartikel (refereegranskat)abstract
- Mass spectrometry has come into its own as an extremely powerful tool for the study of whole proteomes. So why are not more cell biologists embracing it with open arms?
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| 4. |
- Boström, Pontus, 1982, et al.
(författare)
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SNARE proteins mediate fusion between cytosolic lipid droplets and are implicated in insulin sensitivity.
- 2007
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Ingår i: Nature cell biology. - : Springer Science and Business Media LLC. - 1465-7392 .- 1476-4679. ; 9:11, s. 1286-93
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Tidskriftsartikel (refereegranskat)abstract
- The accumulation of cytosolic lipid droplets in muscle and liver cells has been linked to the development of insulin resistance and type 2 diabetes. Such droplets are formed as small structures that increase in size through fusion, a process that is dependent on intact microtubules and the motor protein dynein. Approximately 15% of all droplets are involved in fusion processes at a given time. Here, we show that lipid droplets are associated with proteins involved in fusion processes in the cell: NSF (N-ethylmaleimide-sensitive-factor), alpha-SNAP (soluble NSF attachment protein) and the SNAREs (SNAP receptors), SNAP23 (synaptosomal-associated protein of 23 kDa), syntaxin-5 and VAMP4 (vesicle-associated membrane protein 4). Knockdown of the genes for SNAP23, syntaxin-5 or VAMP4, or microinjection of a dominant-negative mutant of alpha-SNAP, decreases the rate of fusion and the size of the lipid droplets. Thus, the SNARE system seems to have an important role in lipid droplet fusion. We also show that oleic acid treatment decreases the insulin sensitivity of heart muscle cells, and this sensitivity is completely restored by transfection with SNAP23. Thus, SNAP23 might be a link between insulin sensitivity and the inflow of fatty acids to the cell.
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| 5. |
- Gilchrist, Annalyn, et al.
(författare)
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Quantitative proteomics analysis of the secretory pathway.
- 2006
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Ingår i: Cell. - : Elsevier BV. - 0092-8674. ; 127:6, s. 1265-81
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Tidskriftsartikel (refereegranskat)abstract
- We report more than 1400 proteins of the secretory-pathway proteome and provide spatial information on the relative presence of each protein in the rough and smooth ER Golgi cisternae and Golgi-derived COPI vesicles. The data support a role for COPI vesicles in recycling and cisternal maturation, showing that Golgi-resident proteins are present at a higher concentration than secretory cargo. Of the 1400 proteins, 345 were identified as previously uncharacterized. Of these, 230 had their subcellular location deduced by proteomics. This study provides a comprehensive catalog of the ER and Golgi proteomes with insight into their identity and function.
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| 6. |
- Grabenbauer, Markus, et al.
(författare)
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Correlative microscopy and electron tomography of GFP through photooxidation.
- 2005
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Ingår i: Nature methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 2:11, s. 857-62
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a simple correlative photooxidation method that allows for the direct ultrastructural visualization of the green fluorescent protein (GFP) upon illumination. The method, termed GRAB for GFP recognition after bleaching, uses oxygen radicals generated during the GFP bleaching process to photooxidize 3,3'-diaminobenzidine (DAB) into an electron-dense precipitate that can be visualized by routine electron microscopy and electron tomography. The amount of DAB product produced by the GRAB method appears to be linear with the initial fluorescence, and the resulting images are of sufficient quality to reveal detailed spatial information. This is exemplified by the observed intra-Golgi stack and intracisternal distribution of a human Golgi resident glycosylation enzyme, N-acetylgalactosaminyltransferase-2 fused either to enhanced GFP or CFP.
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| 7. |
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| 8. |
- Kartberg, Fredrik, 1978, et al.
(författare)
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Commuting between Golgi cisternae--mind the GAP!
- 2005
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Ingår i: Biochimica et biophysica acta. - : Elsevier BV. - 0006-3002. ; 1744:3, s. 351-63
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Forskningsöversikt (refereegranskat)abstract
- Intracellular transport has remained central to cell biology now for more than 40 years. Despite this, we still lack an overall mechanistic framework that describes transport in different parts of the cell. In the secretory pathway, basic questions, such as how biosynthetic cargo traverses the pathway, are still debated. Historically, emphasis was first put on interpreting function from morphology at the ultrastructural level revealing membrane structures such as the transitional ER, vesicular carriers, vesicular tubular clusters, Golgi cisternae, Golgi stacks and the Golgi ribbon. This emphasis on morphology later switched to biochemistry and yeast genetics yielding many of the key molecular players and their associated functions that we know today. More recently, microscopy studies of living cells incorporating biophysics and system analysis has proven useful and is often used to readdress earlier findings, sometimes with surprising outcomes.
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| 9. |
- Simpson, Jeremy C, et al.
(författare)
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Biogenesis of tubular ER-to-Golgi transport intermediates.
- 2006
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Ingår i: Molecular biology of the cell. - 1059-1524. ; 17:2, s. 723-37
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Tidskriftsartikel (refereegranskat)abstract
- Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.
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| 10. |
- Young, Joanne, et al.
(författare)
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Regulation of microtubule-dependent recycling at the trans-Golgi network by Rab6A and Rab6A'.
- 2005
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Ingår i: Molecular biology of the cell. - 1059-1524. ; 16:1, s. 162-77
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Tidskriftsartikel (refereegranskat)abstract
- The small GTPase rab6A but not the isoform rab6A' has previously been identified as a regulator of the COPI-independent recycling route that carries Golgi-resident proteins and certain toxins from the Golgi to the endoplasmic reticulum (ER). The isoform rab6A' has been implicated in Golgi-to-endosomal recycling. Because rab6A but not A', binds rabkinesin6, this motor protein is proposed to mediate COPI-independent recycling. We show here that both rab6A and rab6A' GTP-restricted mutants promote, with similar efficiency, a microtubule-dependent recycling of Golgi resident glycosylation enzymes upon overexpression. Moreover, we used small interfering RNA mediated down-regulation of rab6A and A' expression and found that reduced levels of rab6 perturbs organization of the Golgi apparatus and delays Golgi-to-ER recycling. Rab6-directed Golgi-to-ER recycling seems to require functional dynactin, as overexpression of p50/dynamitin, or a C-terminal fragment of Bicaudal-D, both known to interact with dynactin inhibit recycling. We further present evidence that rab6-mediated recycling seems to be initiated from the trans-Golgi network. Together, this suggests that a recycling pathway operates at the level of the trans-Golgi linking directly to the ER. This pathway would be the preferred route for both toxins and resident Golgi proteins.
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