scholarly journals The Tip Growth Apparatus of Aspergillus nidulans

2008 ◽  
Vol 19 (4) ◽  
pp. 1439-1449 ◽  
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.

scholarly journals The Role of Microtubules in Rapid Hyphal Tip Growth of Aspergillus nidulans

2005 ◽  
Vol 16 (2) ◽  
pp. 918-926 ◽  
Author(s):  
Tetsuya Horio ◽  
Berl R. Oakley
Keyword(s):  
Growth Rate ◽  
Aspergillus Nidulans ◽  
Tip Growth ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Polarized Growth ◽  
Green Fluorescent ◽  
Tip Cells ◽  
Hyphal Tip

The filamentous fungus Aspergillus nidulans grows by polarized extension of hyphal tips. The actin cytoskeleton is essential for polarized growth, but the role of microtubules has been controversial. To define the role of microtubules in tip growth, we used time-lapse microscopy to measure tip growth rates in germlings of A. nidulans and in multinucleate hyphal tip cells, and we used a green fluorescent protein-α-tubulin fusion to observe the effects of the antimicrotubule agent benomyl. Hyphal tip cells grew ≈5 times faster than binucleate germlings. In germlings, cytoplasmic microtubules disassembled completely in mitosis. In hyphal tip cells, however, microtubules disassembled through most of the cytoplasm in mitosis but persisted in a region near the hyphal tip. The growth rate of hyphal tip cells did not change significantly in mitosis. Benomyl caused rapid disassembly of microtubules in tip cells and a 10× reduction in growth rate. When benomyl was washed out, microtubules assembled quickly and rapid tip growth resumed. These results demonstrate that although microtubules are not strictly required for polarized growth, they are rate-limiting for the growth of hyphal tip cells. These data also reveal that A. nidulans exhibits a remarkable spatial regulation of microtubule disassembly within hyphal tip cells.

Two divergent plasma membrane syntaxin-like SNAREs, nsyn1 and nsyn2, contribute to hyphal tip growth and other developmental processes in Neurospora crassa

2003 ◽  
Vol 40 (3) ◽  
pp. 271-286 ◽  
Author(s):  
Gagan D Gupta ◽  
Stephen J Free ◽  
Natalia N Levina ◽  
Sirkka Keränen ◽  
I.Brent Heath
Keyword(s):  
Plasma Membrane ◽  
Neurospora Crassa ◽  
Tip Growth ◽  
Developmental Processes ◽  
Hyphal Tip Growth ◽  
Hyphal Tip

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

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.

scholarly journals High-throughput live-imaging of embryos in microwell arrays using a modular, inexpensive specimen mounting system

2017 ◽  
Author(s):  
Seth Donoughe ◽  
Chiyoung Kim ◽  
Cassandra G. Extavour
Keyword(s):  
High Throughput ◽  
Fruit Fly ◽  
Time Lapse ◽  
Live Imaging ◽  
Model Systems ◽  
Wide Range ◽  
Amphipod Crustacean ◽  
Live Image ◽  
Time Lapse Imaging ◽  
Annelid Worm

AbstractLive-imaging embryos in a high-throughput manner is essential for shedding light on a wide range of questions in developmental biology, but it is difficult and costly to mount and image embryos in consistent conditions. Here, we present OMMAwell, a simple, reusable device that makes it easy to mount up to hundreds of embryos in arrays of agarose microwells with customizable dimensions and spacing. OMMAwell can be configured to mount specimens for upright or inverted microscopes, and includes a reservoir to hold live-imaging medium to maintain constant moisture and osmolarity of specimens during time-lapse imaging. All device components can be cut from a sheet of acrylic using a laser cutter. Even a novice user will be able to cut the pieces and assemble the device in less than an hour. At the time of writing, the total materials cost is less than five US dollars. We include all device design files in a commonly used format, as well as complete instructions for its fabrication and use. We demonstrate a detailed workflow for designing a custom mold and employing it to simultaneously live-image dozens of embryos at a time for more than five days, using embryos of the cricket Gryllus bimaculatus as an example. Further, we include descriptions, schematics, and design files for molds that can be used with 14 additional vertebrate and invertebrate species, including most major traditional laboratory models and a number of emerging model systems. Molds have been user-tested for embryos including zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), coqui frog (Eleutherodactylus coqui), annelid worm (Capitella teleta), amphipod crustacean (Parhyale hawaiensis), red flour beetle (Tribolium castaneum), and three-banded panther worm (Hofstenia miamia), as well mouse organoids (Mus musculus). Finally, we provide instructions for researchers to customize OMMAwell inserts for embryos or tissues not described herein.Summary StatementThis Techniques and Resources article describes an inexpensive, customizable device for mounting and live-imaging a wide range of tissues and species; complete design files and instructions for assembly are included.

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

2019 ◽  
Author(s):  
Magdalena Marek ◽  
Vincent Vincenzetti ◽  
Sophie G. Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Integrity ◽  
Physiological Role ◽  
Time Lapse ◽  
Vesicular Trafficking ◽  
Protein Oligomerization ◽  
Family Protein ◽  
State Growth ◽  
Eukaryotic Organisms ◽  
Time Lapse Imaging

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.

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

2019 ◽  
Vol 317 (1) ◽  
pp. F124-F132 ◽  
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.

The dynamic behaviour of microtubules and their contributions to hyphal tip growth in Aspergillus nidulans

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1543-1555 ◽  
Author(s):  
Karina Sampson ◽  
I. Brent Heath
Keyword(s):  
Aspergillus Nidulans ◽  
Tip Growth ◽  
Spindle Pole ◽  
Hyphal Tip Growth ◽  
Complex Processes ◽  
Spindle Pole Bodies ◽  
Tubulin Subunit ◽  
Apical Cells ◽  
Tip Extension ◽  
Hyphal Tip

Creating and maintaining cell polarity are complex processes that are not fully understood. Fungal hyphal tip growth is a highly polarized and dynamic process involving both F-actin and microtubules (MTs), but the behaviour and roles of the latter are unclear. To address this issue, MT dynamics and subunit distribution were analysed in a strain of Aspergillus nidulans expressing GFP–α-tubulin. Apical MTs are the most dynamic, the bulk of which move tipwards from multiple subapical spindle pole bodies, the only clear region of microtubule nucleation detected. MTs populate the apex predominantly by elongation at rates about three times faster than tip extension. This polymerization was facilitated by the tipward migration of MT subunits, which generated a tip-high gradient. Subapical regions of apical cells showed variable tubulin subunit distributions, without tipward flow, while subapical cells showed even tubulin subunit distribution and low MT dynamics. Short MTs, of a similar size to those reported in axons, also occasionally slid into the apex. During mitosis in apical cells, MT populations at the tip varied. Cells with less distance between the tip and the first nucleus were more likely to loose normal MT populations and dynamics. Reduced MTs in the tip, during mitosis or after exposure to the MT inhibitor carbendazim (MBC), generally correlated with reduced, but continuing growth and near-normal tip morphology. In contrast, the actin-disrupting agent latrunculin B reduced growth rates much more severely and dramatically distorted tip morphology. These results suggest substantial independence between MTs and hyphal tip growth and a more essential role for F-actin. Among MT-dependent processes possibly contributing to tip growth is the transportation of vesicles. However, preliminary ultrastructural data indicated a lack of direct MT–organelle interactions. It is suggested that the population of dynamic apical MTs enhance migration of the ‘cytomatrix’, thus ensuring that organelles and proteins maintain proximity to the constantly elongating tip.

scholarly journals Deuterium-labeled Raman tracking of glucose accumulation and protein metabolic dynamics in Aspergillus nidulans hyphal tips

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mitsuru Yasuda ◽  
Norio Takeshita ◽  
Shinsuke Shigeto
Keyword(s):  
Aspergillus Nidulans ◽  
Filamentous Fungi ◽  
Raman Band ◽  
Time Lapse ◽  
Raman Imaging ◽  
Host Cells ◽  
Apical Region ◽  
Glucose Accumulation ◽  
Hyphal Tip

AbstractFilamentous fungi grow exclusively at their tips, where many growth-related fungal processes, such as enzyme secretion and invasion into host cells, take place. Hyphal tips are also a site of active metabolism. Understanding metabolic dynamics within the tip region is therefore important for biotechnology and medicine as well as for microbiology and ecology. However, methods that can track metabolic dynamics with sufficient spatial resolution and in a nondestructive manner are highly limited. Here we present time-lapse Raman imaging using a deuterium (D) tracer to study spatiotemporally varying metabolic activity within the hyphal tip of Aspergillus nidulans. By analyzing the carbon–deuterium (C–D) stretching Raman band with spectral deconvolution, we visualize glucose accumulation along the inner edge of the hyphal tip and synthesis of new proteins from the taken-up D-labeled glucose specifically at the central part of the apical region. Our results show that deuterium-labeled Raman imaging offers a broadly applicable platform for the study of metabolic dynamics in filamentous fungi and other relevant microorganisms in vivo.

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