Plant Lipid Bodies Traffic on Actin to Plasmodesmata Motorized by Myosin XIs

Late 19th-century cytologists observed tiny oil drops in shoot parenchyma and seeds, but it was discovered only in 1972 that they were bound by a half unit-membrane. Later, it was found that lipid bodies (LBs) arise from the endoplasmic reticulum. Seeds are known to be packed with static LBs, coated with the LB-specific protein OLEOSIN. As shown here, apices of Populus tremula x P. tremuloides also express OLEOSIN genes and produce potentially mobile LBs. In developing buds, PtOLEOSIN (PtOLE) genes were upregulated, especially PtOLE6, concomitant with LB accumulation. To investigate LB mobility and destinations, we transformed Arabidopsis with PtOLE6-eGFP. We found that PtOLE6-eGFP fusion protein co-localized with Nile Red-stained LBs in all cell types. Moreover, PtOLE6-eGFP-tagged LBs targeted plasmodesmata, identified by the callose marker aniline blue. Pharmacological experiments with brefeldin, cytochalasin D, and oryzalin showed that LB-trafficking requires F-actin, implying involvement of myosin motors. In a triple myosin-XI knockout (xi-k/1/2), transformed with PtOLE6-eGFP, trafficking of PtOLE6-eGFP-tagged LBs was severely impaired, confirming that they move on F-actin, motorized by myosin XIs. The data reveal that LBs and OLEOSINs both function in proliferating apices and buds, and that directional trafficking of LBs to plasmodesmata requires the actomyosin system.

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Cell-specific posttranslational processing of the surfactant-associated protein SP-B

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1993.264.3.l290 ◽

1993 ◽

Vol 264 (3) ◽

pp. L290-L299 ◽

Cited By ~ 4

Author(s):

S. Hawgood ◽

D. Latham ◽

J. Borchelt ◽

D. Damm ◽

T. White ◽

...

Keyword(s):

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Cell Types ◽

Specific Protein ◽

Precursor Protein ◽

Type Ii Cells ◽

Posttranslational Processing ◽

Type Ii ◽

Clara Cells ◽

Ovary Cells

Pulmonary surfactant-associated protein B (SP-B) is a 9-kDa lung-specific protein expressed in alveolar epithelial type II cells and Clara cells. The protein markedly increases the surface activity of phospholipids and is an active component in some surfactants in clinical use. SP-B is produced from a 43-kDa precursor protein by proteolytic cleavage of flanking regions from both the NH2- and COOH-terminal ends of the active protein. In this study we have compared the nature of the posttranslational processing of the SP-B precursor in type II cells and in a heterologous cell line transfected with the SP-B precursor. We found that isolated type II cells produce the 9-kDa form of SP-B from the precursor through a series of intermediates detectable in the cell lysates. In contrast Chinese hamster ovary cells stably transfected with the full-length human SP-B precursor produce the precursor and a 26-kDa intermediate but not the 9-kDa protein. The precursor protein in both cell types is glycosylated with NH2-linked sugars. Our results suggest there is cell specificity in the posttranslational processing of the SP-B precursor.

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Generation of asymmetry during development. Segregation of type-specific proteins in Caulobacter.

The Journal of Cell Biology ◽

10.1083/jcb.81.1.123 ◽

1979 ◽

Vol 81 (1) ◽

pp. 123-136 ◽

Cited By ~ 18

Author(s):

N Agabian ◽

M Evinger ◽

G Parker

Keyword(s):

Cell Cycle ◽

Cell Division ◽

Messenger Rna ◽

Cell Types ◽

Specific Protein ◽

Soluble Proteins ◽

Specific Cell ◽

Daughter Cells ◽

Specific Proteins ◽

Entire Cell

An essential event in developmental processes is the introduction of asymmetry into an otherwise undifferentiated cell population. Cell division in Caulobacter is asymmetric; the progeny cells are structurally different and follow different sequences of development, thus providing a useful model system for the study of differentiation. Because the progeny cells are different from one another, there must be a segregation of morphogenetic and informational components at some time in the cell cycle. We have examined the pattern of specific protein segregation between Caulobacter stalked and swarmer daughter cells, with the rationale that such a progeny analysis would identify both structurally and developmentally important proteins. To complement the study, we have also examined the pattern of protein synthesis during synchronous growth and in various cellular fractions. We show here, for the first time, that the association of proteins with a specific cell type may result not only from their periodicity of synthesis, but also from their pattern of distribution at the time of cell division. Several membrane-associated and soluble proteins are segregated asymmetrically between progeny stalked and swarmer cells. The data further show that a subclass of soluble proteins becomes associated with the membrane of the progeny stalked cells. Therefore, although the principal differentiated cell types possess different synthetic capabilities and characteristic proteins, the asymmetry between progeny stalked and swarmer cells is generated primarily by the preferential association of specific soluble proteins with the membrane of only one daughter cell. The majority of the proteins which exhibit this segregation behavior are synthesized during the entire cell cycle and exhibit relatively long, functional messenger RNA half-lives.

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Arabidopsis Myosin XIK Interacts with the Exocyst Complex to Facilitate Vesicle Tethering during Exocytosis

10.1101/2020.08.18.255984 ◽

2020 ◽

Author(s):

Weiwei Zhang ◽

Lei Huang ◽

Chunhua Zhang ◽

Christopher J. Staiger

Keyword(s):

Mammalian Cells ◽

Secretory Vesicle ◽

Tail Domain ◽

Spatiotemporal Resolution ◽

Dynamic Regulation ◽

Vesicle Tethering ◽

Exocyst Complex ◽

Myosin Xi ◽

Myosin Motors

ABSTRACTMyosin motors are essential players in secretory vesicle trafficking and exocytosis in yeast and mammalian cells; however, similar roles in plants remain a matter for debate, at least for diffusely-growing cells. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) myosin XIK, via its globular tail domain (GTD), participates in the vesicle tethering step of exocytosis through direct interactions with the exocyst complex. Specifically, myosin XIK GTD bound directly to the SEC5B subunit of exocyst in vitro and functional fluorescently-tagged XIK colocalized with multiple exocyst subunits at plasma membrane (PM)-associated stationary foci. Moreover, genetic and pharmacological inhibition of myosin XI activity reduced the frequency and lifetime of stationary exocyst complexes at the PM. By tracking single exocytosis events of cellulose synthase (CESA) complexes (CSCs) with high spatiotemporal resolution imaging and pair-wise colocalization analysis of myosin XIK, exocyst subunits and CESA6, we demonstrated that XIK associates with secretory vesicles earlier than exocyst and is required for the recruitment of exocyst to the PM tethering site. This study reveals an important functional role for myosin XI in secretion and provides new insights about the dynamic regulation of exocytosis in plants.

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Postprandial transfer of colostral extracellular vesicles and their protein and miRNA cargo in neonatal calves

10.21203/rs.2.10032/v1 ◽

2019 ◽

Author(s):

Benedikt Kirchner ◽

Dominik Buschmann ◽

Vijay Paul ◽

Michael W. Pfaffl

Keyword(s):

Extracellular Vesicles ◽

Expression Profiles ◽

Bovine Milk ◽

Cell Types ◽

Specific Protein ◽

In Vivo Experiments ◽

Neonatal Calves ◽

Almost All

Abstract Background Extracellular vesicles (EVs) such as exosomes are key regulators of intercellular communication that can be found in almost all bio fluids. Although studies in the last decade have made great headway in discerning the role of EVs in many physiological and pathophysiological processes, the bioavailability and impact of dietary EVs and their cargo still remain to be elucidated. Due to its widespread consumption and high content of EV-associated microRNAs and proteins, a major focus in this field has been set on EVs in bovine milk and colostrum. Despite promising in vitro studies in recent years that show high resiliency of milk EVs to degradation and uptake of milk EV cargo in a variety of intestinal and blood cell types, in vivo experiments continue to be inconclusive and sometimes outright contradictive. Results To resolve this discrepancy, we assessed the potential postprandial transfer of colostral EVs to the circulation of newborn calves by analysing colostrum-specific protein and miRNAs, including specific isoforms (isomiRs) in cells, EV isolations and unfractionated samples from blood and colostrum. Our findings reveal distinct populations of EVs in colostrum and blood from cows that can be clearly separated by density, particle concentration and protein content (BTN1A1, MFGE8). Postprandial blood samples of calves show a time-dependent increase in EVs that share morphological and protein characteristics of colostral EVs. Analysis of miRNA expression profiles by Next-Generation Sequencing gave a different picture however. Although significant postprandial expression changes could only be detected for calf EV samples, expression profiles show very limited overlap with highly expressed miRNAs in colostral EVs or colostrum in general. Conclusions Taken together our results indicate a selective uptake of membrane-associated protein cargo but not luminal miRNAs from colostral EVs into the circulation of neonatal calves.

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Involvement of cyclic AMP-dependent protein kinases in the signal transduction pathway for interleukin-1

Molecular and Cellular Biology ◽

10.1128/mcb.10.7.3824-3827.1990 ◽

1990 ◽

Vol 10 (7) ◽

pp. 3824-3827

Author(s):

M Chedid ◽

S B Mizel

Keyword(s):

Signal Transduction ◽

Cyclic Amp ◽

Protein Kinases ◽

Gene Transcription ◽

Interleukin 1 ◽

Signal Transduction Pathway ◽

Cell Types ◽

Specific Protein ◽

Transduction Pathway ◽

Dependent Protein

Expression of a highly specific protein inhibitor for cyclic AMP-dependent protein kinases in interleukin-1 (IL-1)-responsive cells blocked IL-1-induced gene transcription that was driven by the kappa immunoglobulin enhancer or the human immunodeficiency virus long terminal repeat. This inhibitor did not affect protein kinase C-mediated gene transcription, suggesting that cyclic AMP-dependent protein kinases are involved in the signal transduction pathway for IL-1 in a number of responsive cell types.

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Phosphoinositide lipids and cell polarity: linking the plasma membrane to the cytocortex

Essays in Biochemistry ◽

10.1042/bse0530015 ◽

2012 ◽

Vol 53 ◽

pp. 15-27 ◽

Cited By ~ 22

Author(s):

Michael P. Krahn ◽

Andreas Wodarz

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Membrane Lipids ◽

Leading Edge ◽

Cell Types ◽

Specific Protein ◽

Molecular Organization ◽

Apical Side ◽

Cell Substrate ◽

Basolateral Side

Many cell types in animals and plants are polarized, which means that the cell is subdivided into functionally and structurally distinct compartments. Epithelial cells, for example, possess an apical side facing a lumen or the outside environment and a basolateral side facing adjacent epithelial cells and the basement membrane. Neurons possess distinct axonal and dendritic compartments with specific functions in sending and receiving signals. Migrating cells form a leading edge that actively engages in pathfinding and cell-substrate attachment, and a trailing edge where such attachments are abandoned. In all of these cases, both the plasma membrane and the cytocortex directly underneath the plasma membrane show differences in their molecular composition and structural organization. In this chapter we will focus on a specific type of membrane lipids, the phosphoinositides, because in polarized cells they show a polarized distribution in the plasma membrane. They furthermore influence the molecular organization of the cytocortex by recruiting specific protein binding partners which are involved in the regulation of the cytoskeleton and in signal transduction cascades that control polarity, growth and cell migration.

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NeuN: a useful neuronal marker for diagnostic histopathology.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.10.8813082 ◽

1996 ◽

Vol 44 (10) ◽

pp. 1167-1171 ◽

Cited By ~ 338

Author(s):

H K Wolf ◽

R Buslei ◽

R Schmidt-Kastner ◽

P K Schmidt-Kastner ◽

T Pietsch ◽

...

Keyword(s):

Neuronal Cell ◽

Cell Types ◽

Specific Protein ◽

Antigen Retrieval ◽

Neuronal Marker ◽

Formalin Fixed Paraffin ◽

Wide Range ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed ◽

Diagnostic Histopathology

The monoclonal antibody A60 specifically recognizes the DNA-binding, neuron-specific protein NeuN, which is present in most neuronal cell types of vertebrates. In this study we demonstrate the potential use of NeuN as a diagnostic neuronal marker using a wide range of formalin-fixed, paraffin-embedded human surgical and autopsy specimens from the central and peripheral nervous system. After microwave antigen retrieval, almost all neuronal populations revealed strong immunoreactivity for NeuN in nuclei, perikarya, and some proximal neuronal processes, whereas more distal axon cylinders and dendritic ramifications were not stained. The stain greatly enhanced the gray matter architecture. NeuN immunoreactivity was not detected in Purkinje cells, most neurons of the internal nuclear layer of the retina, and in sympathetic chain ganglia. We examined nine gangliogliomas and 14 dysembryoplastic neuroepithelial tumors, one ganglioneuroma, and one dysplastic cerebellar gangliocytoma. The neuronal component of all of these lesions showed marked immunoreactivity for NeuN. In addition, NeuN immunoreactivity was focally seen in one of seven medulloblastomas with prominent neuronal differentiation. There was no staining of non-neuronal structures. The results indicate that NeuN immunoreactivity is a sensitive and specific neuronal marker in formalin-fixed, paraffin-embedded tissues, and may be useful in diagnostic histopathology.

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Axl Is Essential for in-vitro Angiogenesis Induced by Vitreous From Patients With Proliferative Diabetic Retinopathy

Frontiers in Medicine ◽

10.3389/fmed.2021.787150 ◽

2021 ◽

Vol 8 ◽

Author(s):

Wenyi Wu ◽

Huizuo Xu ◽

Zhishang Meng ◽

Jianxi Zhu ◽

Siqi Xiong ◽

...

Keyword(s):

Endothelial Cells ◽

Diabetic Retinopathy ◽

Proliferative Diabetic Retinopathy ◽

Tyrosine Kinases ◽

Vascular Endothelial Cells ◽

Specific Inhibitor ◽

Cell Types ◽

Specific Protein ◽

Vascular Endothelial

Proliferative diabetic retinopathy (PDR), characterized mainly with abnormal epiretinal angiogenesis forming fibrovascular membranes (FVMs), threatens vision of people with diabetes; FVMs consist of extracellular matrix and a variety of cell types including vascular endothelial cells. Axl, one of receptor tyrosine kinases, can be activated indirectly by vascular endothelial growth factor-A (VEGF-A) via an intracellular route for promoting angiogenesis. In this study, we revealed that growth arrest-specific protein 6 (Gas6), a specific ligand of Axl, was elevated in vitreous from patients with PDR and that Axl was activated in FVMs from patients with PDR. In addition, we demonstrated that in cultured human retinal microvascular endothelial cells (HRECs), Axl inhibition via suppression of Axl expression with Clustered Regularly Interspaced Short Palindromic Repeats/ CRISPR-associated protein 9 or through inactivation with its specific inhibitor R428 blocked PDR vitreous-induced Akt activation and proliferation of HRECs. Furthermore, PDR vitreous-heightened migration and tube formation of HRECs were also blunted by restraining Axl. These results indicate that in the pathogenesis of PDR, Axl can be activated by Gas6 binding directly and by VEGF-A via an intracellular route indirectly, suggesting that Axl plays a pivotal role in the development of PDR and that Axl inhibition shows a bright promise for PDR therapy.

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Synaptosomes: New Vesicles for Neuronal Mitochondrial Transplantation

10.21203/rs.3.rs-89848/v1 ◽

2020 ◽

Author(s):

Pasquale Picone ◽

Gaetana Porcelli ◽

Celeste Caruso Bavisotto ◽

Domenico Nuzzo ◽

Giacoma Galizzi ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Neuronal Cells ◽

Nile Red ◽

Critical Factor ◽

Specific Protein ◽

Mitochondrial Proteins ◽

Hexokinase Ii ◽

Protein Protein Interaction ◽

First Time ◽

Fusion Ability

Abstract Background: Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, subcellular fraction of isolated synaptic terminal that contain mitochondria, as mitochondrial delivery systems. Results: Synaptosomes preparation was validated by the presence of Synaptophysin and PSD95. Syn aptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/mL. Nile Red or CTX-FITCH labeled synaptosomes were internalized in LAN5 recipient cells by a mechanism involving specific protein-protein interaction, as demonstrated by loss of fusion ability after trypsin treatment and using different cell lines. The loading and release ability of the synaptosomes was proved by the presence of curcumin both into synaptosomes and LAN5 cells. The vitality of mitochondria transferred by Synaptosomes was demonstrated by the presence of Opa1, Fis1 and TOM40 mitochondrial proteins and JC-1 measurements. Further, synaptosomes deliver vital mitochondria into the cytoplasm of neuronal cells as demonstrated by microscopic images, increase of TOM 40, cytochrome c, Hexokinase II mitochondrial proteins, and presence of rat mitochondrial DNA. Finally, by using synaptosomes as vehicle, healthy mitochondria restored mitochondrial function in cells containing rotenone or CCCp damaged mitochondria. Conclusions: Taken together these results suggest that synaptosomes can be a natural vehicle for the delivery of molecules and organelles to neuronal cells. Further, replacement of affected mitochondria with healthy ones could be a potential therapy for the treatment of neuronal mitochondrial dysfunction-related diseases.

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Proximity labeling of protein complexes and cell-type-specific organellar proteomes in Arabidopsis enabled by TurboID

eLife ◽

10.7554/elife.47864 ◽

2019 ◽

Vol 8 ◽

Cited By ~ 22

Author(s):

Andrea Mair ◽

Shou-Ling Xu ◽

Tess C Branon ◽

Alice Y Ting ◽

Dominique C Bergmann

Keyword(s):

Transcription Factor ◽

Protein Interactions ◽

Protein Complexes ◽

Cell Types ◽

Specific Protein ◽

Cell Type ◽

Subcellular Compartment ◽

Cellular Processes ◽

Highly Active

Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

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