Martina Gentzsch, PhD

Research Methods Collaborations Biosketch Contact
Martina Small

Dr. Martina Gentzsch, Assistant Professor, Department of Cell Biology and Physiology; Director, CFTR Correction Core


Research Interest

Processing and Trafficking Defect of ∆F508 CFTR

We study intracellular trafficking of the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR), which is mutated in cystic fibrosis. The most common mutation of CFTR, ∆F508, results in a misassembled protein that is retained at the Endoplasmic Reticulum (Fig. 1) but can escape and proceed to the plasma membrane by addition of small-molecule correctors (Fig. 2). However, rescued ∆F508 disappears rapidly from the cell surface and is subjected to lysosomal degradation, while wild-type CFTR is recycled back to the plasma membrane. The goal of our research is to elucidate a means to restore proper localization and stability of ∆F508 CFTR in human airway epithelia. Specifically, we aim to identify therapeutic targets and pharmacological treatments that allow modulation of the cell surface density and stability of mutant CFTR proteins in cystic fibrosis.

Mechanism of Regulation of ENaC by CFTR

In cystic fibrosis bronchial epithelia, deficient CFTR channel activity and the absence of CFTR-dependent inhibition of the epithelial sodium channel (ENaC) lead to dehydration of airway surfaces. While it has been established that mutations in CFTR result in defective regulation of ENaC and consequent sodium hyperabsorption in cystic fibrosis airways, the detailed mechanism by which CFTR mediates inhibition of ENaC remains unclear. ENaC is known to be stimulated by limited proteolysis of the extracellular domains and we have demonstrated recently that CFTR impedes proteolytic processing of ENaC (Fig. 3).

Membrane Trafficking Mechanisms that Govern Channel Density at the Surface

Individually overexpressed ENaC subunits (α, β, γ) reside, in the absence of the other subunits, exclusively at the Endoplasmic Reticulum, however, co-expression of all three subunits results in surface expression and co-localization with CFTR at the plasma membrane (Fig. 4A,B). Insertion of an extracellular tag into the γ-subunit of ENaC and labeling with an antibody on intact cells allows confirmation of the surface localization and also permits monitoring of intracellular trafficking of ENaC (Fig. 4C). Similar to CFTR, ENaC is internalized from the plasma membrane and transfers to endocytic compartments. Currently, we are aiming to understand the membrane trafficking mechanisms that govern channel density of CFTR and ENaC at the apical surface.

Figure 1_Page

Figure 1.Intracellular trafficking of CFTR. A. Cartoon depicting intracellular CFTR trafficking routes. B. DeltaF508 CFTR has a processing and trafficking defect. In differentiated primary human airway epithelial cells, adenovirally overexpressed wild-type CFTR localized to the apical membrane, while deltaF508 was present only intracellularly, as shown by confocal microscopy of CFTR fluorescent labeling (bar=10µM; CFTR is shown in green, cilia that were labeled with anti-tubulin antibody in red and nuclei in blue). See full size image...

Figure 2.Corrector rescue of mutant CFTR in primary human airway epithelial cultures (HAE). A. Normal (NL) or CF HAE cells were treated with DMSO (Vehicle) or 5µM VX809 for 48 hours and then processed for immunoprecipitation and Western blotting. B.Isc tracing of NL and CF HAE cells after treatment with vehicle or >5µM VX809. See full-size image...

Figure 3.CFTR impedes proteolysis of ENaC. Expression of increasing amounts of CFTR (shown in red) diminishes appearance of matriptase-induced proteolytic fragments of α-ENaC (shown in green). CFTR, ENaC and matriptase were co-expressed in Xenopus oocytes and detected by Western blot analysis using an infrared imaging system. Mechanism of Regulation of ENac by CFTR. See full-size image...

Figure 4.Intracellular trafficking of ENaC and CFTR. A. Individually expressed ENaC subunits localize to the ER. ENaC subunits were expressed individually in BHK-21 cells and immunostained. B. Co-expressed ENaC subunits reach the cell surface, where they co-localize with CFTR. α-, β-, and γ-ENaC were coexpressed together with CFTR. C. Extracellular epitope tags of γ-ENaC (coexpressed with α- and β-ENaC) and CFTR were labeled at the cell surface and labeled proteins were visualized after a 30 min chase. See full-size image...

 

  • Research Methods

  • Analysis of Processing and Modulation of CFTR and ENaC by Biochemical Methods
    i.e. Western Blot, ELISA, Immunoprecipitation, Surface Biotinylation
  • Modulation (correction and potentiation) of mutant CFTR in primary human airway cultures
  • Analysis of CFTR and ENaC in intestinal biopsies
  • Design of New Methods to Track CFTR Rescue and Stability
  • Primary Cell Cultures of Human, Mouse, and Pig Epithelial Airways
  • Cell-based Assays in Microtiter Plates to Evaluate Mutant CFTR Rescue
  • Transient Transfection and Generation of Stable Cell Lines
  • Expression in Xenopus oocytes
  • Confocal Immunofluorescence and Live Cell Microscopy
  • Pulse-Chase Experiments
  • Endocytosis and Recycling Assays
  • Protein Analysis of Nasal, Bronchial, and Intestinal Tissue
  • Sample Prep for Mass Spectrometry-based Proteomics
  • Analysis of Protein Glycosylation and Secretion
  • Viral Protein Overexpression
  • Silencing (retroviral and lentiviral vectors)
  • Ion Channel Electrophysiology

 

Collaborations

Monroe J. Stutts, PhD: ENaC-CFTR interaction and regulation of ENaC by CFTR.
Nikolay V. Dokholyan: Structure of ENaC.
John R. Riordan, PhD: CFTR: structure, function and processing.
Robert Tarran, PhD: CFTR in apical macromolecular complexes and modulation of CFTR trafficking. Regulation of ENaC.
Raymond J. Pickles, PhD: Trafficking of parainfluenza virus vector expressed CFTR and ENaC.
Wanda O’Neal, PhD: Generation of adeno- and retroviral vectors for expression of extracellularly tagged CFTR and ENaC.
Scott H. Randell, PhD: Analysis of CFTR and ENaC in primary human airway epithelial cultures and lung tissue.

Biographical Sketch

Martina Gentzsch, PhD, Assistant Professor of Cell and Developmental Biology

Education

1997 Ph.D. Cell Biology, SUMMA CUM LAUDE
University of Regensburg, Germany
1993 M.S. Cell Biology, Biochemistry, Genetics, Organic Chemistry,
University of Regensburg, Germany
1991 B.S. Genetics, Physics, Chemistry, Botany, University of Regensburg, Germany

Professional Experience

2012-present: Research Assistant Professor of Cell Biology and Physiology
University of North Carolina at Chapel Hill, NC
2012-present: Director, CFTR Correction Core
Cystic Fibrosis/Pulmonary Research & Treatment Center
2005-2012: Research Assistant Professor of Cell and Developmental Biology
Cystic Fibrosis/Pulmonary Research & Treatment Center
University of North Carolina, Chapel Hill, NC
2001-2005: Research Associate
Department of Biochemistry/Molecular Biology, Mayo Clinic, Scottsdale, AZ
1998-2001: Postdoctoral Research Fellow
Laboratory of J.R. Riordan, Mayo Clinic College of Medicine, Scottsdale, AZ
1997-1998: Postdoctoral Fellow
Laboratory of W. Tanner, Institute of Cell Biology, University of Regensburg, Germany

Other Experience

2012 Chair: Symposium "CFTR: The Conductance Regulator", 26th Annual North American Cystic Fibrosis Conference, Orlando, FL
2011 Chair: Symposium "Proteases and Lung Disease", 34th ECFS Conference, Hamburg, Germany
2010 Chair: Workshop "CFTR and Cell Biology", 33rd ECFS Conference, Valencia, Spain
2010 Chair: Symposium “WT and ∆F508del CFTR trafficking: learning the rules of the road"
33rd ECFS Basic Science Conference, Carcavelos, Portugal
2008-2010 Organizer of the UNC Cystic Fibrosis Center “Epithelial Cell Biology Seminar Series”
2008 Chair: Workshop Session "CFTR – The Biology & Pathology",
2nd FEBS Special Meeting on ABC Proteins, ABC2008, Innsbruck, Austria
2007 Chair: Symposia "CFTR Structure" and "Membrane Traffic of CFTR",
Chair: Discussion Group "Endo/Exocytic Regulation of CFTR",
Basic Science Conference, European Cystic Fibrosis Society, Tavira, Portugal
2006 Chair: Workshop "CFTR 2006", 20th NACFC, Denver, CO
2005 Intensive Course Quantitative Fluorescence Microscopy, MDIBL, Salisbury Cove, ME
1998 Course in Cell Culture and Tissue Engineering, Regensburg, Germany (Dr. W Minuth)
1997 Research Internship, MIT, Cambridge, MA, USA (Dr. S. Sanders)
1993 Research Internship, Glaxo Institute for Molecular Biology, Geneva, Switzerland

Memberships and Honors

2010 Profiled author of “paper of the week”, Journal of Biological Chemistry
2009 UNC CF-Center Star Heel Award
2008 Member of the American Physiological Society
2005 Member of the American Society for Cell Biology
2003 Young Investigator Award, 4th FEBS Advanced Lecture Course on
ATP-Binding Cassette Proteins, Gosau, Austria
1998-2000 Research Stipend from the German Research Association DFG
1997 Culture award for Ph.D. thesis, E.ON group (Europe’s largest energy provider)
1997 Ph.D. Honors graduation: summa cum laude

Selected Invited Presentations

Gentzsch M (2012) Getting the CFTR ion channel back on the right track: Restoring trafficking of CFTR in cystic fibrosis airways. Department of Medical Biochemistry, University of Gothenburg, Sweden
Gentzsch M (2011) Processing Trafficking and Stability Defect of ∆F508 CFTR. 11th Symposium Mukoviskidose e.V. "Current Aspects in the Pathophysiology of Cystic Fibrosis", Geseke, Germany
Gentzsch M (2011) Proteases and Ion Transport Regulation: Proteolysis Controls ENaC Activity in Airways. 34th ECFS Conference, Hamburg, Germany
Gentzsch M (2010) Interaction of ENaC and CFTR. ECFS Basic Science Conference, Carcavelos, Portugal
Gentzsch M (2010) Endocytic Cycling of CFTR and ∆F508 CFTR. 33rd ECFS Conference, Valencia, Spain
Cholon DM, O'Neal WK, Riordan JR, Gentzsch M (2008) Modulation of apical stability of rescued ∆F508 CFTR in primary human airway epithelial cells. 22nd NACFC, CFF, Orlando, FL. Pediatr Pulmonol, Suppl 30:203. (Presented by DMC)
Gentzsch M, Mengos A, O'Neal WK, Riordan JR (2007) Processing and turnover of CFTR in highly differentiated cultures of human airway epithelia. European Cystic Fibrosis Conference - New Frontiers in Basic Science of Cystic Fibrosis, Tavira, Algarve, Portugal.
Gentzsch M, Choudhury A, Chang XB, Pagano RE, Riordan JR (2006) Misassembled mutant F508 ∆CFTR in the distal secretory pathway alters cellular cholesterol and glycosphingolipid trafficking. 20th NACFC, CFF, Denver, CO. Pediatr Pulmonol, Suppl 29:212.
Gentzsch M, Mengos A, Chang XB, Hou YX, Cui L, Aleksandrov A, Riordan JR (2005) Neither calnexin binding nor other carbohydrate-mediated interactions are responsible for ∆F508 CFTR retention at the ER. 19th NACFC, CFF, Baltimore, MD, Pediatr Pulmonol, Suppl 28:194.
Gentzsch, M (2004) Role of Oligosaccharide-mediated ER Chaperone Interactions in Processing and Trafficking of Wild-type and ∆F508 CFTR. CFF Conference, Williamsburg, VA.
Gentzsch M, Cui L, Mengos A, Chang XB, Chen JH, Riordan JR (2003) Subcellular localization of PDZ-binding chloride channels CFTR and ClC-3B. 4th Consensus Meeting Towards Validation of CFTR Gene Expression and Functional Assays, Cystic Fibrosis European Network, Cascais, Portugal.
Gentzsch M, Chang, XB, Wu Y, Cui L, Riordan JR (2003) CFTR trafficking in the endocytic pathway. 26th European Cystic Fibrosis Conference, European Cystic Fibrosis Society, Belfast, Northern Ireland, Journal of Cystic Fibrosis, Vol 2, Suppl 1:S52.
Gentzsch M, Aleksandrov A, Aleksandrov L, Riordan JR (2001) Functional analysis of truncated CFTR proteins reveals the C‑terminal boundary of Nucleotide Binding Domain 2. 15th NACFC, CFF, Orlando, FL, Pediatr Pulmonol Supp. 22:180-181.
Gentzsch M, Aleksandrov A, Riordan JR (2001) C-terminal sequences impact processing and ion channel activity of CFTR. 3rd FEBS Advanced Lecture Course "ATP-Binding Cassette (ABC) Proteins", Gosau, Austria.
Gentzsch M, Riordan JR (2000) Impact of the C-terminus on the stability and maturation of nascent CFTR. XIIIth International CF Congress, International CF Association (ICFA), Stockholm, Sweden.
Gentzsch M, Hammerle M, Chen JS, Mengos A, Jensen T. Chang XB, Riordan JR (2000) Early life of the CFTR in the cell. Forefronts in Nephrology Meeting: Regulation of Membrane proteins, International Society of Nephrology, Hayama-machi, Kanagawa, Japan, Kidney Int 60(2):401.

Funding Sources

Cystic Fibrosis Foundation (CFF), National Institutes of Health (NIH), Else Kröner-Fresenius-Stiftung

Publications

Please see PubMed feed in the right-hand column for links to current publications.

 

Lab Personnel

Martina Small

Martina Gentzsch, PhD, Assistant Professor of Cell Biology and Physiology

Deborah_Small

Deborah Cholon, PhD, Postdoctoral Research Associate

Jennifer Small 2013

Jennifer Guimbellot, MD, PhD, Pediatric Pulmonary Fellow

 

 

 

 

 

 

 

 

 

Contact Information

6109A Thurston-Bowles Bldg.
Campus Box #7248
The University of North Carolina at Chapel Hill
Chapel Hill, NC 27599
Phone:(919) 843-4716
Fax:(919)966-5178
E-mail: gentzsch@med.unc.edu