skip to main content

Attention:

The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 5:00 PM ET until 11:00 PM ET on Friday, June 21 due to maintenance. We apologize for the inconvenience.


Title: Hydrodynamic dissection of Stentor coeruleus in a microfluidic cross junction
Stentor coeruleus , a single-cell ciliated protozoan, is a model organism for wound healing and regeneration studies. Despite Stentor 's large size (up to 2 mm in extended state), microdissection of Stentor remains challenging. In this work, we describe a hydrodynamic cell splitter, consisting of a microfluidic cross junction, capable of splitting Stentor cells in a non-contact manner at a high throughput of ∼500 cells per minute under continuous operation. Introduction of asymmetry in the flow field at the cross junction leads to asymmetric splitting of the cells to generate cell fragments as small as ∼8.5 times the original cell size. Characterization of cell fragment viability shows reduced 5-day survival as fragment size decreases and as the extent of hydrodynamic stress imposed on the fragments increases. Our results suggest that cell fragment size and composition, as well as mechanical stress, play important roles in the long-term repair of Stentor cells and warrant further investigations. Nevertheless, the hydrodynamic splitter can be useful for studying phenomena immediately after cell splitting, such as the closure of wounds in the plasma membrane which occurs on the order of 100–1000 seconds in Stentor .  more » « less
Award ID(s):
1938109
NSF-PAR ID:
10393874
Author(s) / Creator(s):
; ; ; ; ;
Date Published:
Journal Name:
Lab on a Chip
Volume:
22
Issue:
18
ISSN:
1473-0197
Page Range / eLocation ID:
3508 to 3520
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Abstract

    Ciliates are powerful unicellular model organisms that have been used to elucidate fundamental biological processes. However, the high motility of ciliates presents a major challenge in studies using live-cell microscopy and microsurgery. While various immobilization methods have been developed, they are physiologically disruptive to the cell and incompatible with microscopy and/or microsurgery. Here, we describe a Simple Microfluidic Operating Room for the Examination and Surgery ofStentor coeruleus(SMORES). SMORES uses Quake valve-based microfluidics to trap, compress, and perform surgery onStentoras our model ciliate. Compared with previous methods, immobilization by physical compression in SMORES is more effective and uniform. The mean velocity of compressed cells is 24 times less than that of uncompressed cells. The compression is minimally disruptive to the cell and is easily applied or removed using a 3D-printed pressure rig. We demonstrate cell immobilization for up to 2 h without sacrificing cell viability. SMORES is compatible with confocal microscopy and is capable of media exchange for pharmacokinetic studies. Finally, the modular design of SMORES allows laser ablation or mechanical dissection of a cell into many cell fragments at once. These capabilities are expected to enable biological studies previously impossible in ciliates and other motile species.

     
    more » « less
  2. Brennan, Richard Gerald (Ed.)
    ABSTRACT <p>Microbial extracellular proteins and metabolites provide valuable information concerning how microbes adapt to changing environments. In cyanobacteria, dynamic acclimation strategies involve a variety of regulatory mechanisms, being ferric uptake regulator proteins as key players in this process. In the nitrogen-fixing cyanobacterium<italic>Anabaena</italic>sp. strain PCC 7120, FurC (PerR) is a global regulator that modulates the peroxide response and several genes involved in photosynthesis and nitrogen metabolism. To investigate the possible role of FurC in shaping the extracellular environment of<italic>Anabaena</italic>, the analysis of the extracellular metabolites and proteins of a<italic>furC</italic>-overexpressing variant was compared to that of the wild-type strain. There were 96 differentially abundant proteins, 78 of which were found for the first time in the extracellular fraction of<italic>Anabaena</italic>. While these proteins belong to different functional categories, most of them are predicted to be secreted or have a peripheral location. Several stress-related proteins, including PrxA, flavodoxin, and the Dps homolog All1173, accumulated in the exoproteome of<italic>furC</italic>-overexpressing cells, while decreased levels of FurA and a subset of membrane proteins, including several export proteins and<italic>amiC</italic>gene products, responsible for nanopore formation, were detected. Direct repression by FurC of some of those genes, including<italic>amiC1</italic>and<italic>amiC2,</italic>could account for odd septal nanopore formation and impaired intercellular molecular transfer observed in the<italic>furC</italic>-overexpressing variant. Assessment of the exometabolome from both strains revealed the release of two peptidoglycan fragments in<italic>furC</italic>-overexpressing cells, namely 1,6-anhydro-N-acetyl-β-D-muramic acid (anhydroMurNAc) and its associated disaccharide (β-D-GlcNAc-(1-4)-anhydroMurNAc), suggesting alterations in peptidoglycan breakdown and recycling.</p><sec><title>IMPORTANCE

    Cyanobacteria are ubiquitous photosynthetic prokaryotes that can adapt to environmental stresses by modulating their extracellular contents. Measurements of the organization and composition of the extracellular milieu provide useful information about cyanobacterial adaptive processes, which can potentially lead to biomimetic approaches to stabilizing biological systems to adverse conditions.Anabaenasp. strain PCC 7120 is a multicellular, nitrogen-fixing cyanobacterium whose intercellular molecular exchange is mediated by septal junctions that traverse the septal peptidoglycan through nanopores. FurC (PerR) is an essential transcriptional regulator inAnabaena, which modulates the response to several stresses. Here, we show thatfurC-overexpressing cells result in a modified exoproteome and the release of peptidoglycan fragments. Phenotypically, important alterations in nanopore formation and cell-to-cell communication were observed. Our results expand the roles of FurC to the modulation of cell-wall biogenesis and recycling, as well as in intercellular molecular transfer.

     
    more » « less
  3.  
    more » « less
  4. SUMMARY

    Translating ribosome affinity purification (TRAP) utilizes transgenic plants expressing a ribosomal protein fused to a tag for affinity co‐purification of ribosomes and the mRNAs that they are translating. This population of actively translated mRNAs (translatome) can be interrogated by quantitative PCR or RNA sequencing. Condition‐ or cell‐specific promoters can be utilized to isolate the translatome of specific cell types, at different growth stages and/or in response to environmental variables. While advantageous for revealing differential expression, this approach may not provide sufficient sensitivity when activity of the condition/cell‐specific promoter is weak, when ribosome turnover is low in the cells of interest, or when the targeted cells are ephemeral. In these situations, expressing tagged ribosomes under the control of these specific promoters may not yield sufficient polysomes for downstream analysis. Here, we describe a new TRAP system that employs two transgenes: One is constitutively expressed and encodes a ribosomal protein fused to one fragment of a split green fluorescent protein (GFP); the second is controlled by a stimulus‐specific promoter and encodes the second GFP fragment fused to an affinity purification tag. In cells where both transgenes are active, the purification tag is attached to ribosomes by bi‐molecular folding and assembly of the split GFP fragments. This approach provides increased sensitivity and better temporal resolution because it labels pre‐existing ribosomes and does not depend on rapid ribosome turnover. We describe the optimization and key parameters of this system, and then apply it to a plant–pathogen interaction in which spatial and temporal resolution are difficult to achieve with current technologies.

     
    more » « less
  5. Abstract

    Habitat fragmentation remains a major focus of research by ecologists decades after being put forward as a threat to the integrity of ecosystems. While studies have documented myriad biotic changes in fragmented landscapes, including the local extinction of species from fragments, the demographic mechanisms underlying these extinctions are rarely known. However, many of them—especially in lowland tropical forests—are thought to be driven by one of two mechanisms: (1) reduced recruitment in fragments resulting from changes in the diversity or abundance of pollinators and seed dispersers or (2) increased rates of individual mortality in fragments due to dramatically altered abiotic conditions, especially near fragment edges. Unfortunately, there have been few tests of these potential mechanisms due to the paucity of long‐term and comprehensive demographic data collected in both forest fragments and continuous forest sites. Here we report 11 years (1998–2009) of demographic data from populations of the Amazonian understory herbHeliconia acuminata(LC Rich.) found at Brazil's Biological Dynamics of Forest Fragments Project (BDFFP). The data set comprises >66,000 plant × year records of 8586 plants, including 3464 seedlings established after the first census. Seven populations were in experimentally isolated fragments (one in each of four 1‐ha fragments and one in each of three 10‐ha fragments), with the remaining six populations in continuous forest. Each population was in a 50 × 100 m permanent plot, with the distance between plots ranging from 500 m to 60 km. The plants in each plot were censused annually, at which time we recorded, identified, marked, and measured new seedlings, identified any previously marked plants that died, and recorded the size of surviving individuals. Each plot was also surveyed four to five times during the flowering season to identify reproductive plants and record the number of inflorescences each produced. These data have been used to investigate topics ranging from the way fragmentation‐related reductions in germination influence population dynamics to statistical methods for analyzing reproductive rates. This breadth of prior use reflects the value of these data to future researchers. In addition to analyses of plant responses to habitat fragmentation, these data can be used to address fundamental questions in plant demography and the evolutionary ecology of tropical plants and to develop and test demographic models and tools. Though we welcome opportunities to collaborate with interested users, there are no restrictions on the use of this data set. However, we do request that those using the data for teaching or research purposes inform us of how they are doing so and cite this paper and the data archive when appropriate. Any publication using the data must also include a BDFFP Technical Series Number in the Acknowledgments. Authors can request this series number upon the acceptance of their article by contacting the BDFFP's Scientific Coordinator or E. M. Bruna.

     
    more » « less