Sterol flow between the plasma membrane and the endosome is regulated by the LAM family protein Ltc1

AbstractSterols are crucial components of biological membranes that help maintain membrane integrity and regulate various processes such as endocytosis, protein oligomerization and signaling. Although synthetized in the ER, sterols are at highest concentrations at the plasma membrane (PM) in all eukaryotic organisms. Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. While D4H is detected at the PM during steady-state growth, inhibition of Arp2/3-dependent F-actin assembly unexpectedly promotes the reversible re-localization of the probe to internal sterol rich compartments (STRIC), as shown by correlative light-electron microscopy. Time-lapse imaging identifies STRIC as a late secretory, endosomal compartment labelled by the synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi. Instead, it depends on Ltc1, a LAM/StARkin-family protein that localizes to ER-PM contact sites. In ltc1Δ, sterols over-accumulate at the PM, which forms extended ER-interacting invaginations, indicating that sterol transfer by Ltc1 contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking components but requires Arp2/3 activity, suggesting a novel physiological role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the retrograde and anterograde flow of sterols in the cell.

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Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition

The Journal of Cell Biology ◽

10.1083/jcb.202001147 ◽

2020 ◽

Vol 219 (6) ◽

Cited By ~ 3

Author(s):

Magdalena Marek ◽

Vincent Vincenzetti ◽

Sophie G. Martin

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Biological Membranes ◽

Time Lapse ◽

Vesicular Trafficking ◽

Actin Assembly ◽

Contact Sites ◽

Family Protein

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell.

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Tripartite-motif family protein 35-28 regulates microglia development by preventing necrotic death of microglial precursors in zebrafish

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.012043 ◽

2020 ◽

Vol 295 (26) ◽

pp. 8846-8856

Author(s):

Tao Yu ◽

Haoyue Kuang ◽

Jiahao Chen ◽

Xi Lin ◽

Yi Wu ◽

...

Keyword(s):

Regulatory Protein ◽

Neutrophil Infiltration ◽

Time Lapse ◽

Membrane Rupture ◽

Family Protein ◽

Tripartite Motif ◽

The Central Nervous System ◽

Time Lapse Imaging ◽

Identification And Characterization

Microglia are tissue-resident macrophages in the central nervous system (CNS) that play essential roles in the regulation of CNS development and homeostasis. Yet, the genetic networks governing microglia development remain incompletely defined. Here, we report the identification and characterization of a microglia-defective zebrafish mutant wulonghkz12 (wulhkz12) isolated from an ethylnitrosourea (ENU)-based genetic screen. We show that wulhkz12 mutants harbors a missense point mutation in the gene region encoding the PRY/SPRY domain of the tripartite-motif family protein 35-28 (trim35-28) gene. Time-lapse imaging revealed that the loss of Trim35-28 function causes lytic necrosis of microglial precursors/peripheral macrophages, as indicated by cytoplasmic swelling and membrane rupture of these precursors and accompanied by neutrophil infiltration and systemic inflammation. Intriguingly, the lytic necrosis of microglial precursors in trim35-28–deficient mutants appeared to depend neither on the canonical pyroptotic nor necroptotic pathways, as inhibition of the key component in each pathway could not rescue the microglia phenotype in trim35-28–deficient mutants. Finally, results from tissue-specific rescue experiments suggested that Trim35-28 acts cell-autonomously in the survival of microglial precursors. Taken together, the findings of our study reveal Trim35-28 as a regulatory protein essential for microglia development.

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EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

10.1101/224162 ◽

2017 ◽

Author(s):

Chi-Lun Chang ◽

Yu-Ju Chen ◽

Jen Liou

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Live Cell Imaging ◽

Cell Imaging ◽

Time Lapse ◽

Cellular Functions ◽

Binding Ability ◽

Spatiotemporal Regulation ◽

Time Lapse Imaging

AbstractThe endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.SummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

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A versatile aquaporin-2 cell system for quantitative temporal expression and live cell imaging

AJP Renal Physiology ◽

10.1152/ajprenal.00150.2019 ◽

2019 ◽

Vol 317 (1) ◽

pp. F124-F132 ◽

Cited By ~ 2

Author(s):

Mikkel R. Holst ◽

Lene N. Nejsum

Keyword(s):

Plasma Membrane ◽

Cell Migration ◽

Fluorescent Protein ◽

Time Lapse ◽

Cell System ◽

Urine Concentration ◽

Epithelial Morphogenesis ◽

Expression Levels ◽

Aquaporin 2 ◽

Time Lapse Imaging

Aquaporin-2 (AQP2) fine tunes urine concentration in response to the antidiuretic hormone vasopressin. In addition, AQP2 has been suggested to promote cell migration and epithelial morphogenesis. A cell system allowing temporal and quantitative control of expression levels of AQP2 and phospho-mimicking mutants has been missing, as has a system allowing expression of fluorescently tagged AQP2 for time-lapse imaging. In the present study, we generated and validated a Flp-In T-REx Madin-Darby canine kidney cell system for temporal and quantitative control of AQP2 and phospho-mimicking mutants. We verified that expression levels can be temporally and quantitatively controlled and that AQP2 translocated to the plasma membrane in response to elevated cAMP, which also induced S256 phosphorylation. The phospho-mimicking mutants AQP2-S256A and AQP2-S256D localized as previously described, primarily intracellular and to the plasma membrane, respectively. Induction of AQP2 expression in combination with transient, low expression of enhanced green fluorescent protein-tagged AQP2 enabled expression without aggregation and correct translocation in response to elevated cAMP. Interestingly, time-lapse imaging revealed AQP2-containing tubulating endosomes and that tubulation significantly decreased 30 min after cAMP elevation. This was mirrored by the phospho-mimicking mutants AQP2-S256A and AQP2-S256D, where AQP2-S256A-containing endosomes tubulated, whereas AQP2-S256D-containing endosomes did not. Thus, this cell system enables a multitude of cell-based assays warranted to provide deeper insights into the mechanisms of AQP2 regulation and effects on cell migration and epithelial morphogenesis.

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Role of turgor pressure in endocytosis in fission yeast

Molecular Biology of the Cell ◽

10.1091/mbc.e13-10-0618 ◽

2014 ◽

Vol 25 (5) ◽

pp. 679-687 ◽

Cited By ~ 55

Author(s):

Roshni Basu ◽

Emilia Laura Munteanu ◽

Fred Chang

Keyword(s):

Cell Wall ◽

Plasma Membrane ◽

Fission Yeast ◽

Turgor Pressure ◽

Time Lapse ◽

Actin Assembly ◽

Latrunculin A ◽

The Media ◽

Time Lapse Imaging

Yeast and other walled cells possess high internal turgor pressure that allows them to grow and survive in the environment. This turgor pressure, however, may oppose the invagination of the plasma membrane needed for endocytosis. Here we study the effects of turgor pressure on endocytosis in the fission yeast Schizosaccharomyces pombe by time-lapse imaging of individual endocytic sites. Decreasing effective turgor pressure by addition of sorbitol to the media significantly accelerates early steps in the endocytic process before actin assembly and membrane ingression but does not affect the velocity or depth of ingression of the endocytic pit in wild-type cells. Sorbitol also rescues endocytic ingression defects of certain endocytic mutants and of cells treated with a low dose of the actin inhibitor latrunculin A. Endocytosis proceeds after removal of the cell wall, suggesting that the cell wall does not contribute mechanically to this process. These studies suggest that endocytosis is governed by a mechanical balance between local actin-dependent inward forces and opposing forces from high internal turgor pressure on the plasma membrane.

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The Tip Growth Apparatus of Aspergillus nidulans

Molecular Biology of the Cell ◽

10.1091/mbc.e07-05-0464 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1439-1449 ◽

Cited By ~ 205

Author(s):

Naimeh Taheri-Talesh ◽

Tetsuya Horio ◽

Lidia Araujo-Bazán ◽

Xiaowei Dou ◽

Eduardo A. Espeso ◽

...

Keyword(s):

Plasma Membrane ◽

Aspergillus Nidulans ◽

Fusion Proteins ◽

Tip Growth ◽

Time Lapse ◽

Live Imaging ◽

Medical Importance ◽

Cytoskeletal Elements ◽

Time Lapse Imaging ◽

Hyphal Tip

Hyphal tip growth in fungi is important because of the economic and medical importance of fungi, and because it may be a useful model for polarized growth in other organisms. We have investigated the central questions of the roles of cytoskeletal elements and of the precise sites of exocytosis and endocytosis at the growing hyphal tip by using the model fungus Aspergillus nidulans. Time-lapse imaging of fluorescent fusion proteins reveals a remarkably dynamic, but highly structured, tip growth apparatus. Live imaging of SYNA, a synaptobrevin homologue, and SECC, an exocyst component, reveals that vesicles accumulate in the Spitzenkörper (apical body) and fuse with the plasma membrane at the extreme apex of the hypha. SYNA is recycled from the plasma membrane by endocytosis at a collar of endocytic patches, 1–2 μm behind the apex of the hypha, that moves forward as the tip grows. Exocytosis and endocytosis are thus spatially coupled. Inhibitor studies, in combination with observations of fluorescent fusion proteins, reveal that actin functions in exocytosis and endocytosis at the tip and in holding the tip growth apparatus together. Microtubules are important for delivering vesicles to the tip area and for holding the tip growth apparatus in position.

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Cleavage of Human Embryos: Options and Diversity

Acta Naturae ◽

10.32607/20758251-2016-8-3-88-96 ◽

2016 ◽

Vol 8 (3) ◽

pp. 88-96

Author(s):

Yu. K. Doronin ◽

I. V. Senechkin ◽

L. V. Hilkevich ◽

M. A. Kurcer

Keyword(s):

Time Lapse ◽

Reproductive Technologies ◽

Human Embryos ◽

Imaging Data ◽

Noninvasive Methods ◽

Topographic Features ◽

Time Parameters ◽

Embryo Cleavage ◽

Time Lapse Imaging ◽

Developmental Dynamics

In order to estimate the diversity of embryo cleavage relatives to embryo progress (blastocyst formation), time-lapse imaging data of preimplantation human embryo development were used. This retrospective study is focused on the topographic features and time parameters of the cleavages, with particular emphasis on the lengths of cleavage cycles and the genealogy of blastomeres in 2- to 8-cell human embryos. We have found that all 4-cell human embryos have four developmental variants that are based on the sequence of appearance and orientation of cleavage planes during embryo cleavage from 2 to 4 blastomeres. Each variant of cleavage shows a strong correlation with further developmental dynamics of the embryos (different cleavage cycle characteristics as well as lengths of blastomere cycles). An analysis of the sequence of human blastomere divisions allowed us to postulate that the effects of zygotic determinants are eliminated as a result of cleavage, and that, thereafter, blastomeres acquire the ability of own syntheses, regulation, polarization, formation of functional contacts, and, finally, of specific differentiation. This data on the early development of human embryos obtained using noninvasive methods complements and extend our understanding of the embryogenesis of eutherian mammals and may be applied in the practice of reproductive technologies.

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