Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics
Abstract
Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression with single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.
- Authors:
-
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
- Publication Date:
- Research Org.:
- Pacific Northwest National Laboratory (PNNL), Richland, WA (United States)
- Sponsoring Org.:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- OSTI Identifier:
- 1226407
- Report Number(s):
- PNNL-SA-109268
Journal ID: ISSN 2045-2322; 48672; KP1704020
- Grant/Contract Number:
- AC05-76RL01830; 66382
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Scientific Reports
- Additional Journal Information:
- Journal Volume: 5; Journal Issue: 16111; Journal ID: ISSN 2045-2322
- Publisher:
- Nature Publishing Group
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 59 BASIC BIOLOGICAL SCIENCES; microchannel; high-throughput; cell; polarized growth; microfluidic; Neurospora crassa; Environmental Molecular Sciences Laboratory; fungal biology; lab-on-a-chip
Citation Formats
Geng, Tao, Bredeweg, Erin L., Szymanski, Craig J., Liu, Bingwen, Baker, Scott E., Orr, Galya, Evans, James E., and Kelly, Ryan T. Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics. United States: N. p., 2015.
Web. doi:10.1038/srep16111.
Geng, Tao, Bredeweg, Erin L., Szymanski, Craig J., Liu, Bingwen, Baker, Scott E., Orr, Galya, Evans, James E., & Kelly, Ryan T. Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics. United States. https://doi.org/10.1038/srep16111
Geng, Tao, Bredeweg, Erin L., Szymanski, Craig J., Liu, Bingwen, Baker, Scott E., Orr, Galya, Evans, James E., and Kelly, Ryan T. Wed .
"Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics". United States. https://doi.org/10.1038/srep16111. https://www.osti.gov/servlets/purl/1226407.
@article{osti_1226407,
title = {Compartmentalized microchannel array for high-throughput analysis of single cell polarized growth and dynamics},
author = {Geng, Tao and Bredeweg, Erin L. and Szymanski, Craig J. and Liu, Bingwen and Baker, Scott E. and Orr, Galya and Evans, James E. and Kelly, Ryan T.},
abstractNote = {Here, interrogating polarized growth is technologically challenging due to extensive cellular branching and uncontrollable environmental conditions in conventional assays. Here we present a robust and high-performance microfluidic system that enables observations of polarized growth with enhanced temporal and spatial control over prolonged periods. The system has built-in tunability and versatility to accommodate a variety of science applications requiring precisely controlled environments. Using the model filamentous fungus, Neurospora crassa, this microfluidic system enabled direct visualization and analysis of cellular heterogeneity in a clonal fungal cell population, nuclear distribution and dynamics at the subhyphal level, and quantitative dynamics of gene expression with single hyphal compartment resolution in response to carbon source starvation and exchange experiments. Although the microfluidic device is demonstrated on filamentous fungi, our technology is immediately extensible to a wide array of other biosystems that exhibit similar polarized cell growth with applications ranging from bioenergy production to human health.},
doi = {10.1038/srep16111},
journal = {Scientific Reports},
number = 16111,
volume = 5,
place = {United States},
year = {Wed Nov 04 00:00:00 EST 2015},
month = {Wed Nov 04 00:00:00 EST 2015}
}
Web of Science
Works referenced in this record:
Single-molecule imaging of NGF axonal transport in microfluidic devices
journal, January 2010
- Zhang, Kai; Osakada, Yasuko; Vrljic, Marija
- Lab on a Chip, Vol. 10, Issue 19
Microfluidic Platforms for Single-Cell Analysis
journal, July 2010
- Zare, Richard N.; Kim, Samuel
- Annual Review of Biomedical Engineering, Vol. 12, Issue 1
Transcriptional response to glucose starvation and functional analysis of a glucose transporter of Neurospora crassa
journal, December 2004
- Xie, Xin; Wilkinson, Heather H.; Correa, Alejandro
- Fungal Genetics and Biology, Vol. 41, Issue 12
Microfluidic Platforms for Single-Cell Analysis
journal, July 2010
- Zare, Richard N.; Kim, Samuel
- Annual Review of Biomedical Engineering, Vol. 12, Issue 1
How does a hypha grow? The biophysics of pressurized growth in fungi
journal, June 2011
- Lew, Roger R.
- Nature Reviews Microbiology, Vol. 9, Issue 7
Morphology and Growth Kinetics of Hyphae of Differentiated and Undifferentiated Mycelia of Neurospora crassa
journal, December 1975
- Steele, G. C.; Trinci, A. P. J.
- Journal of General Microbiology, Vol. 91, Issue 2
Probing bacterial–fungal interactions at the single cell level
journal, January 2014
- Stanley, Claire E.; Stöckli, Martina; van Swaay, Dirk
- Integr. Biol., Vol. 6, Issue 10
How does a hypha grow? The biophysics of pressurized growth in fungi
journal, June 2011
- Lew, Roger R.
- Nature Reviews Microbiology, Vol. 9, Issue 7
Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells
journal, January 2009
- Faley, Shannon L.; Copland, Mhairi; Wlodkowic, Donald
- Lab on a Chip, Vol. 9, Issue 18
Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform
journal, March 2012
- Lee, S. S.; Vizcarra, I. A.; Huberts, D. H. E. W.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 13
Polarity in filamentous fungi: establishment, maintenance and new axes
journal, December 2002
- Momany, Michelle
- Current Opinion in Microbiology, Vol. 5, Issue 6
Fungal genome sequencing: basic biology to biotechnology
text, January 2015
- Sharma, Krishna Kant
- Taylor & Francis
Growth and Differentiation of Neurospora hyphae
journal, October 1959
- Zalokar, Marko
- American Journal of Botany, Vol. 46, Issue 8
Fungal genome sequencing: basic biology to biotechnology
journal, February 2015
- Sharma, Krishna Kant
- Critical Reviews in Biotechnology
Microfluidic single cell analysis: from promise to practice
journal, August 2012
- Lecault, Véronique; White, Adam K.; Singhal, Anupam
- Current Opinion in Chemical Biology, Vol. 16, Issue 3-4
Nuclear Dynamics, Mitosis, and the Cytoskeleton during the Early Stages of Colony Initiation in Neurospora crassa
journal, March 2010
- Roca, M. Gabriela; Kuo, Hsiao-Che; Lichius, Alexander
- Eukaryotic Cell, Vol. 9, Issue 8
Microfluidic devices for measuring gene network dynamics in single cells
journal, August 2009
- Bennett, Matthew R.; Hasty, Jeff
- Nature Reviews Genetics, Vol. 10, Issue 9
Tracking lineages of single cells in lines using a microfluidic device
journal, October 2009
- Rowat, A. C.; Bird, J. C.; Agresti, J. J.
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 43
An agar gel membrane-PDMS hybrid microfluidic device for long term single cell dynamic study
journal, January 2010
- Wong, Ieong; Atsumi, Shota; Huang, Wei-Chih
- Lab on a Chip, Vol. 10, Issue 20
Microfluidic devices for measuring gene network dynamics in single cells
journal, August 2009
- Bennett, Matthew R.; Hasty, Jeff
- Nature Reviews Genetics, Vol. 10, Issue 9
Tracking lineages of single cells in lines using a microfluidic device
journal, October 2009
- Rowat, A. C.; Bird, J. C.; Agresti, J. J.
- Proceedings of the National Academy of Sciences, Vol. 106, Issue 43
Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography
journal, April 2000
- Unger, M. A.
- Science, Vol. 288, Issue 5463
Growth and Differentiation of Neurospora Hyphae
journal, October 1959
- Zalokar, Marko
- American Journal of Botany, Vol. 46, Issue 8
Observing Single Cell NF-κB Dynamics under Stimulant Concentration Gradient
journal, January 2012
- Awwad, Yousef; Geng, Tao; Baldwin, Albert S.
- Analytical Chemistry, Vol. 84, Issue 3
Whole lifespan microscopic observation of budding yeast aging through a microfluidic dissection platform
text, January 2012
- Lee, S. S.; Vizcarra, I. A.; Huberts, D. H. E. W.
- National Academy of Sciences
Multilayer microfluidic devices created from a single photomask
journal, January 2013
- Kelly, Ryan T.; Sheen, Allison M.; Jambovane, Sachin
- RSC Advances, Vol. 3, Issue 43
Fungi Use Efficient Algorithms for the Exploration of Microfluidic Networks
journal, August 2006
- Hanson, Kristi L.; Nicolau, Dan V.; Filipponi, Luisa
- Small, Vol. 2, Issue 10
Morphology and Growth Kinetics of Hyphae of Differentiated and Undifferentiated Mycelia of Neurospora crassa
journal, December 1975
- Steele, G. C.; Trinci, A. P. J.
- Journal of General Microbiology, Vol. 91, Issue 2
Nuclear dynamics in a fungal chimera
journal, July 2013
- Roper, M.; Simonin, A.; Hickey, P. C.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 32
Microfluidic single cell arrays to interrogate signalling dynamics of individual, patient-derived hematopoietic stem cells
journal, January 2009
- Faley, Shannon L.; Copland, Mhairi; Wlodkowic, Donald
- Lab on a Chip, Vol. 9, Issue 18
MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast
journal, February 2007
- Paliwal, Saurabh; Iglesias, Pablo A.; Campbell, Kyle
- Nature, Vol. 446, Issue 7131
TipChip: a modular, MEMS-based platform for experimentation and phenotyping of tip-growing cells
journal, January 2013
- Agudelo, Carlos G.; Sanati Nezhad, Amir; Ghanbari, Mahmood
- The Plant Journal, Vol. 73, Issue 6
Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis
journal, January 2010
- Du, Jing; Li, Sijin; Zhao, Huimin
- Molecular BioSystems, Vol. 6, Issue 11
Cytoplasmic Bulk Flow Propels Nuclei in Mature Hyphae of Neurospora crassa
journal, August 2009
- Ramos-García, Silvia L.; Roberson, Robert W.; Freitag, Michael
- Eukaryotic Cell, Vol. 8, Issue 12
A Study of the Kinetics of Hyphal Extension and Branch Initiation of Fungal Mycelia
journal, March 1974
- Trinci, A. P. J.
- Journal of General Microbiology, Vol. 81, Issue 1
Transcriptional comparison of the filamentous fungus Neurospora crassa growing on three major monosaccharides D-glucose, D-xylose and L-arabinose
journal, January 2014
- Li, Jingen; Lin, Liangcai; Li, Huiyan
- Biotechnology for Biofuels, Vol. 7, Issue 1
The tube Method of Measuring the Growth rate of Neurospora
journal, December 1943
- Ryan, Francis J.; Beadle, G. W.; Tatum, E. L.
- American Journal of Botany, Vol. 30, Issue 10
A Generalizable, Tunable Microfluidic Platform for Delivering Fast Temporally Varying Chemical Signals to Probe Single-Cell Response Dynamics
journal, October 2014
- Chingozha, Loice; Zhan, Mei; Zhu, Cheng
- Analytical Chemistry, Vol. 86, Issue 20
A Generalizable, Tunable Microfluidic Platform for Delivering Fast Temporally Varying Chemical Signals to Probe Single-Cell Response Dynamics
journal, October 2014
- Chingozha, Loice; Zhan, Mei; Zhu, Cheng
- Analytical Chemistry, Vol. 86, Issue 20
Fungal genome sequencing: basic biology to biotechnology
text, January 2015
- Sharma, Krishna Kant
- Taylor & Francis
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii
journal, March 2001
- Alberti-Segui, Christine; Dietrich, Fred; Altmann-Jöhl, Regula
- Journal of Cell Science, Vol. 114, Issue 5
Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat
journal, July 2005
- Balagadde, F. K.
- Science, Vol. 309, Issue 5731
The Tube Method of Measuring the Growth Rate of Neurospora
journal, December 1943
- Ryan, Francis J.; Beadle, G. W.; Tatum, E. L.
- American Journal of Botany, Vol. 30, Issue 10
The architecture of polarized cell growth: The unique status of elongating plant cells
journal, May 2003
- Balu?ka, Franti?ek; Wojtaszek, Przemys?aw; Volkmann, Dieter
- BioEssays, Vol. 25, Issue 6
Microfluidic Local Perfusion Chambers for the Visualization and Manipulation of Synapses
journal, April 2010
- Taylor, Anne M.; Dieterich, Daniela C.; Ito, Hiroshi T.
- Neuron, Vol. 66, Issue 1
Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat
journal, July 2005
- Balagadde, F. K.
- Science, Vol. 309, Issue 5731
The architecture of polarized cell growth: The unique status of elongating plant cells
journal, May 2003
- Balu?ka, Franti?ek; Wojtaszek, Przemys?aw; Volkmann, Dieter
- BioEssays, Vol. 25, Issue 6
Single cell transcriptomics of neighboring hyphae of Aspergillus niger
journal, January 2011
- de Bekker, Charissa; Bruning, Oskar; Jonker, Martijs J.
- Genome Biology, Vol. 12, Issue 8
Single cell transcriptomics of neighboring hyphae of Aspergillus niger
journal, January 2011
- de Bekker, Charissa; Bruning, Oskar; Jonker, Martijs J.
- Genome Biology, Vol. 12, Issue 8
Discovery and characterization of novel d-xylose-specific transporters from Neurospora crassa and Pichia stipitis
journal, January 2010
- Du, Jing; Li, Sijin; Zhao, Huimin
- Molecular BioSystems, Vol. 6, Issue 11
Observing Single Cell NF-κB Dynamics under Stimulant Concentration Gradient
journal, January 2012
- Awwad, Yousef; Geng, Tao; Baldwin, Albert S.
- Analytical Chemistry, Vol. 84, Issue 3
High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays
journal, May 2011
- Lecault, Véronique; VanInsberghe, Michael; Sekulovic, Sanja
- Nature Methods, Vol. 8, Issue 7
Microfluidic Local Perfusion Chambers for the Visualization and Manipulation of Synapses
journal, April 2010
- Taylor, Anne M.; Dieterich, Daniela C.; Ito, Hiroshi T.
- Neuron, Vol. 66, Issue 1
A microfluidic chemostat for experiments with bacterial and yeast cells
journal, August 2005
- Groisman, Alex; Lobo, Caroline; Cho, HoJung
- Nature Methods, Vol. 2, Issue 9
MAPK-mediated bimodal gene expression and adaptive gradient sensing in yeast
journal, February 2007
- Paliwal, Saurabh; Iglesias, Pablo A.; Campbell, Kyle
- Nature, Vol. 446, Issue 7131
Nuclear dynamics in a fungal chimera
journal, July 2013
- Roper, M.; Simonin, A.; Hickey, P. C.
- Proceedings of the National Academy of Sciences, Vol. 110, Issue 32
Mass flow and velocity profiles in Neurospora hyphae: partial plug flow dominates intra-hyphal transport
journal, August 2013
- Abadeh, A.; Lew, R. R.
- Microbiology, Vol. 159, Issue Pt_11
Microfluidic single cell analysis: from promise to practice
journal, August 2012
- Lecault, Véronique; White, Adam K.; Singhal, Anupam
- Current Opinion in Chemical Biology, Vol. 16, Issue 3-4
Microfluidic chemostat for measuring single cell dynamics in bacteria
journal, January 2013
- Long, Zhicheng; Nugent, Eileen; Javer, Avelino
- Lab on a Chip, Vol. 13, Issue 5
Histone modification analysis by chromatin immunoprecipitation from a low number of cells on a microfluidic platform
journal, January 2011
- Geng, Tao; Bao, Ning; Litt, Michael D.
- Lab on a Chip, Vol. 11, Issue 17
Histone modification analysis by chromatin immunoprecipitation from a low number of cells on a microfluidic platform
journal, January 2011
- Geng, Tao; Bao, Ning; Litt, Michael D.
- Lab on a Chip, Vol. 11, Issue 17
Long-term model predictive control of gene expression at the population and single-cell levels
journal, August 2012
- Uhlendorf, J.; Miermont, A.; Delaveau, T.
- Proceedings of the National Academy of Sciences, Vol. 109, Issue 35
Mapping the Growth of Fungal Hyphae: Orthogonal Cell Wall Expansion during Tip Growth and the Role of Turgor
journal, November 2000
- Bartnicki-Garcia, Salomon; Bracker, Charles E.; Gierz, Gerhard
- Biophysical Journal, Vol. 79, Issue 5
TipChip: a modular, MEMS-based platform for experimentation and phenotyping of tip-growing cells
journal, January 2013
- Agudelo, Carlos G.; Sanati Nezhad, Amir; Ghanbari, Mahmood
- The Plant Journal, Vol. 73, Issue 6
Fungi Use Efficient Algorithms for the Exploration of Microfluidic Networks
journal, August 2006
- Hanson, Kristi L.; Nicolau, Dan V.; Filipponi, Luisa
- Small, Vol. 2, Issue 10
Neurospora: a model of model microbes
journal, May 2002
- Davis, Rowland H.; Perkins, David D.
- Nature Reviews Genetics, Vol. 3, Issue 5
Probing the growth dynamics of Neurospora crassa with microfluidic structures
journal, June 2011
- Held, Marie; Edwards, Clive; Nicolau, Dan V.
- Fungal Biology, Vol. 115, Issue 6
Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies : Hyphal heterogeneity is maintained by septal closure
journal, November 2012
- Bleichrodt, Robert-Jan; van Veluw, G. Jerre; Recter, Brand
- Molecular Microbiology, Vol. 86, Issue 6
Observing Single Cell NF-κB Dynamics under Stimulant Concentration Gradient
text, January 2012
- Yousef, Awwad,; Tao, Geng,; S., Baldwin, Albert
- The University of North Carolina at Chapel Hill University Libraries
A microfluidic chemostat for experiments with bacterial and yeast cells
journal, August 2005
- Groisman, Alex; Lobo, Caroline; Cho, HoJung
- Nature Methods, Vol. 2, Issue 9
Monolithic Microfabricated Valves and Pumps by Multilayer Soft Lithography
journal, April 2000
- Unger, M. A.
- Science, Vol. 288, Issue 5463
High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays
journal, May 2011
- Lecault, Véronique; VanInsberghe, Michael; Sekulovic, Sanja
- Nature Methods, Vol. 8, Issue 7
Cytoplasmic dynein is required to oppose the force that moves nuclei towards the hyphal tip in the filamentous ascomycete Ashbya gossypii
journal, March 2001
- Alberti-Segui, Christine; Dietrich, Fred; Altmann-Jöhl, Regula
- Journal of Cell Science, Vol. 114, Issue 5
Works referencing / citing this record:
Advances in Microfluidics‐Based Technologies for Single Cell Culture
journal, April 2019
- García Alonso, Daniel; Yu, Mengchao; Qu, Haijun
- Advanced Biosystems, Vol. 3, Issue 11
Intracellular mechanisms of fungal space searching in microenvironments
journal, June 2019
- Held, Marie; Kašpar, Ondřej; Edwards, Clive
- Proceedings of the National Academy of Sciences, Vol. 116, Issue 27
An elastomeric micropillar platform for the study of protrusive forces in hyphal invasion
journal, January 2017
- Tayagui, Ayelen; Sun, Yiling; Collings, David A.
- Lab Chip, Vol. 17, Issue 21
Increasing access to microfluidics for studying fungi and other branched biological structures
journal, June 2019
- Millet, Larry J.; Aufrecht, Jayde; Labbé, Jessy
- Fungal Biology and Biotechnology, Vol. 6, Issue 1
Intracellular mechanisms of fungal space searching in microenvironments
journal, June 2019
- Held, Marie; Kašpar, Ondřej; Edwards, Clive
- Proceedings of the National Academy of Sciences, Vol. 116, Issue 27
Increasing access to microfluidics for studying fungi and other branched biological structures
journal, June 2019
- Millet, Larry J.; Aufrecht, Jayde; Labbé, Jessy
- Fungal Biology and Biotechnology, Vol. 6, Issue 1