Visualization of Lipid Droplet Composition by Direct Organelle Mass Spectrometry

Horn, Patrick J.; Ledbetter, Nicole R.; James, Christopher N.; Hoffman, William D.; Case, Charlene R.; Verbeck, Guido F.; Chapman, Kent D.

doi:10.1074/jbc.m110.186353

Cited by 72 publications

(73 citation statements)

References 35 publications

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“…2 C and F-H). Because validation of Nile red staining in the meristem tissues by mass spectrometry is difficult because of technique limitations, we applied another commonly used lipid dye, Bodipy 493/503 (47), in the same condition. Our results showed that the signal intensity of lipid droplets stained by Bodipy dye was reduced significantly in rpl4d and tirT mutants, similar to the result from Nile red staining (Fig.…”

Section: Rpl4 Mutation Resulted In Reduced Lipid Accumulation In Merimentioning

confidence: 99%

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

Sun

Hicks

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The vacuole is the most prominent compartment in plant cells and is important for ion and protein storage. In our effort to search for key regulators in the plant vacuole sorting pathway, ribosomal large subunit 4 (rpl4d) was identified as a translational mutant defective in both vacuole trafficking and normal development. Polysome profiling of the rpl4d mutant showed reduction in polysome-bound mRNA compared with wild-type, but no significant change in the general mRNA distribution pattern. Ribsomal profiling data indicated that genes in the lipid metabolism pathways were translationally down-regulated in the rpl4d mutant. Live imaging studies by Nile red staining suggested that both polar and nonpolar lipid accumulation was reduced in meristem tissues of rpl4d mutants. Pharmacological evidence showed that sterol and sphingolipid biosynthetic inhibitors can phenocopy the defects of the rpl4d mutant, including an altered vacuole trafficking pattern. Genetic evidence from lipid biosynthetic mutants indicates that alteration in the metabolism of either sterol or sphingolipid biosynthesis resulted in vacuole trafficking defects, similar to the rpl4d mutant. Tissue-specific complementation with key enzymes from lipid biosynthesis pathways can partially rescue both vacuole trafficking and auxin-related developmental defects in the rpl4d mutant. These results indicate that lipid metabolism modulates auxin-mediated tissue differentiation and endomembrane trafficking pathways downstream of ribosomal protein function.

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Section: Rpl4 Mutation Resulted In Reduced Lipid Accumulation In Merimentioning

confidence: 99%

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

Sun

Hicks

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…To address these questions, technological advances are needed to isolate speckles either biochemically or microscopically, which can then be analyzed using the available highly sensitive proteomic methods. Recently, single lipid droplets from cytoplasm were isolated by combining visualization and microphase separation to analyze their composition (Horn et al, 2011). A similar approach might be useful in analyzing the composition of speckles.…”

Section: Conclusion and Perspectivementioning

confidence: 99%

Localization and Dynamics of Nuclear Speckles in Plants

et al. 2011

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The generation of mature mRNAs from most genes (about 80%-90%) in autotrophic eukaryotes requires the removal of noncoding sequences (introns) and splicing of the coding regions (exons; Labadorf et al., 2010). During splicing in some precursor messenger RNAs (pre-mRNAs), the same splice sites are always used, referred to as constitutive splicing (CS), resulting in a single transcript from a gene. However, from many pre-mRNAs, multiple mature mRNAs are generated from a single gene by alternative splicing (AS), where different combinations of splice sites are used. Both CS and AS are critical to the proper expression of intron-containing genes. Recent transcriptome-wide analysis of AS using high-throughput sequencing indicates that pre-mRNAs from up to 42% of introncontaining genes in Arabidopsis (Arabidopsis thaliana; Filichkin et al., 2010) and about 48% in rice (Oryza sativa; Lu et al., 2010) are alternatively spliced, whereas about 95% of human genes are alternatively spliced (Pan et al., 2008). In addition to pre-mRNAs, some primary microRNAs (pri-miRNAs) are also subject to CS and AS (Hirsch et al., 2006;Szarzynska et al., 2009;Mica et al., 2010). AS increases the protein-coding capacity of a genome and generates functionally different proteins from the same gene (Reddy, 2007). AS results in protein isoforms with loss or gain of function and altered subcellular localization, protein stability, and/or posttranslational modifications. Furthermore, AS plays an important role in gene regulation through regulated unproductive splicing and translation, leading to RNA degradation by mRNA surveillance mechanisms, differential recruitment of mRNAs to ribosomes, or translatability of splice variants (Kurihara et al., 2009;Licatalosi and Darnell, 2010;Palusa and Reddy, 2010). Numerous spliceosomal proteins either promote or suppress splicing by interacting with splicing regulatory elements on the pre-mRNAs. In recent years, the localization and dynamics of some splicing regulators in plants have been analyzed using a variety of approaches. This review summarizes the current status of research in this area, with emphasis on RNAbinding proteins (RBPs) that are involved in splicing regulation, discusses open questions, and presents some approaches to address these questions. SPLICING REGULATORSpre-mRNAs splicing takes place cotranscriptionally in the spliceosome, a large multicomponent complex composed of small nuclear RNAs (snRNAs) and about 170 proteins, many of which are involved in the regulation of splicing (Wahl et al., 2009;Valadkhan and Jaladat, 2010). The composition of the human and yeast spliceosome has been analyzed in great detail (Wahl et al., 2009); however, information on plant splicing has been scarce, as no in vitro assembly of a functional plant spliceosome has been possible to date. A detailed search for Arabidopsis orthologs of the human spliceosome proteome revealed that most splicing factors are present in the Arabidopsis genome, indicating a similar complexity (Barta et al., 2011). Interestingly, m...

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“…Perhaps future comparisons of transcriptional profi les between oil palm fruit and seeds (two adjacent tissue systems rich in lipid droplets, one without and one with oleosins), with an eye toward lipid-droplet biogenesis, may provide insights into the generalized and/or unique machinery involved in TAG packaging in plants. These studies will likely be complemented by proteomics analysis of LDs as well as emerging techniques for analyzing the lipid composition (i.e., lipidome) of individual LDs ( 112,113 ).…”

Section: Approaches To Identify Novel Proteins Involved In Ld Biogenementioning

confidence: 99%

Biogenesis and functions of lipid droplets in plants

Chapman

Dyer

Mullen

2012

Journal of Lipid Research

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327

162

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Journal of Lipid Research Volume 53, 2012 215developing seeds and other plant tissues that contribute to the synthesis of TAGs, although the relative contributions of these alternative pathways to TAG accumulation may vary depending on the tissue and/or species ( 1 ).There also has been a growing appreciation in the past few years that the compartmentation of neutral lipids in LDs of plants extends well beyond their role as simply static depots for carbon storage in seeds and, consequently, there is renewed interest in the cellular ontogeny and dynamics of this organelle. For instance, LDs are observed in nearly all cell types in plants, and although the biogenesis of LDs in nonseed tissues is still poorly understood, we now know that they are involved in many unique processes, such as stress response, pathogen resistance, and hormone metabolism. Furthermore, there are highly specialized roles for LDs in anther development, wherein LDs contribute signifi cantly to the formation of the hydrophobic barrier of the pollen coat as tapetal tissues undergo programmed cell death. Interestingly, this process is somewhat similar to the specialized role that LDs play in the formation of the hydrophobic barrier that comprises the outer layer of mammalian skin.Overall, knowledge of LD function in both seed and nonseed tissues has been greatly enhanced by efforts to characterize the major proteins that specifi cally associate with these organelles, namely oleosins, caleosins, and sterol dehydrogenases (steroleosins) ( Fig. 1 ). Here, after a brief description of LD biogenesis in plant cells, we review the functional properties of these major LD-associated proteins and discuss how they compare with the properties of their known or potential counterparts in yeasts and mammals. We also describe the specialized role of LDs in pollen coat formation and how this process has some The seeds of plants store signifi cant amounts of neutral lipids, namely triacylglycerols (TAGs), in cytosolic lipid droplets (LDs), most of which are subsequently mobilized immediately after germination in order to fuel the growth and development of the seedling prior to photosynthetic establishment. In developing seeds, TAGs are assembled in the endoplasmic reticulum (ER) from acyl-CoAs and glycerol by the conserved "Kennedy" pathway that operates in all eukaryotes. Recently, however, additional acylCoA-independent reactions have been identifi ed in

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Visualization of Lipid Droplet Composition by Direct Organelle Mass Spectrometry

Cited by 72 publications

References 35 publications

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

Arabidopsis ribosomal proteins control vacuole trafficking and developmental programs through the regulation of lipid metabolism

Localization and Dynamics of Nuclear Speckles in Plants

Biogenesis and functions of lipid droplets in plants

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