Aracaju, Sergipe, Brazil

Federal University of Sergipe Department of Physics

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EMBO Workshop on Ca Signals in the Cell Nucleus August 20-23, 1998 EMBO Workshop on Signal Transduction-Mediated Regulation of Nuclear Transport August 11-14, 2001

Introduction Nuclear genes determine structure and function in eukaryotes. Their activity is regulated by extra- and intracellular signals which travel from cytosolic to nucleosolic compartments. Most of them use the nuclear pore complexes (NPCs) (at the nuclear envelope, NE, see Laskey, 1998) which regulate the transported cargo (transcription factors, mRNA, etc.) . Since 1990, patch-clamp (e.g., Matzke et al., 1990; Mazzanti et al., 1990) has confirmed classical microelectrode experiments (see refs. below) supporting the idea that NPCs also regulate ion fluxes (reviewed in Loewenstein et al., 1966). NPC ion channel behavior is viewed under the macromolecule-conducting ion channel paradigm ( Bustamante et al., 1995a, see below). These experiments validate patch-clamp as a method for the assessment of nuclear signaling and, thus for the understanding of the mechanisms of control of gene activity and expression.. Thus, they are of relevance to cloning and gene therapy.

NPC Structure

On the right is shown the NPC model from Aebi's group in Basel. The figure is taken from Panté & Aebi, 1994 (with permission). The putative central plug or transporter, containing the famed p62 complexes, is drawn in fuzzy color to indicate the controversy on whether it forms part of the NPC (see below). Cytoplasmic filaments are represented by the vertical, rod-like structures. Nucleoplasmic filaments, on the other side, converge to form basket-like structures. The plug is recognized as an electron-dense material under EM. The material is less frequently seen when macromolecular transport substrates are added. This suggests that the plug is not part of the NPC but material in transit caught during specimen fixation (thus the fuzzy color in the figure). With some exceptions, NPC plugging and macromolecular transport seems to depend on Ca2+(see below) and on RanGTP (see illustrations from the Max Planck Society and The Scientist). Recent advances in proteomics has uncovered the complexities of NPC gating (e.g., Rout & Aitchison, 2001 - full paper with excellent pictures).

Occam's Razor

NPC Ion Channel Behavior & The Molecular Coulter Counter Concept We are investigating NPC structure and function with patch-clamp, confocal, atomic force microscopy (AFM) and, transmission and field-emission electron microscopies (TEM and FESEM). Our results demonstrate that due to their poor electrical charge carrier properties, macromolecules (e.g., transcription factors and DNAs, RNAs) and other particles reduce NPC channel conductance (e.g., Bustamante et al., 1995a). This macromolecule-conducting channel paradigm (see animation below) has been recently applied to the measurement of nucleic acids (e.g., Kasianowicz et al., 1996; Hanss et al., 1998; Akeson et al., 1999; Lubensky & Nelson, 1999; Meller et al., 1999). As macromolecular size increases, the pore becomes plugged and there is no ion flow. This effect is analogous to that of cells in a Coulter counter (e.g., Bezrukov et al., 1994 ; Bezrukov & Kasianowicz, 1997; Merzlyak et al., 1999). For a review on the molecular Coulter counter, see Bezrukov (2000). Fig 2 of Daneholt (1997) illustrates an RNP particle (high molecular weight RNA associated with proteins) being exported (with permission). The study by Kiseleva et al. (1998) with field emission SEM demonstrates the interactions between mRNA and NPCs. Our paradigm is shown below (see Bustamante et al., 1995a). Note that plugging of the NPC diffusional channel was proposed on the basis of fluorescence microscopy observations (see page 350 of Reiner Peters' review Peters, 1986; see also Keminer & Peters, 1999). Mitochondrial ion channels show plugging similar to what we believe is the plugging of NPC ion channel activity (press here for some references on mitochondrial channel). Endoplasmic reticulum (ER) ribosomal channels also showed channel plugging by proteins (see Simon et al., 1989; Simon & Blobel, 1991, 1992). Finally, plant thylakoid membrane channels stop ion flow during protein translocation (e.g. Teter & Theg, 1998).

Selected References (the pros and cons)

Fluorescence Microscopy Showing Neglibible Monoatomic Ion Gradient Across The NE

Giovannardi et al., 1994; O'Malley, 1994; Meyer et al., 1995;
Stehno-Bittel et al., 1995b; Gerasimenko et al., 1996; Brown et al., 1997; Genka et al., 1999

Fluorescence Microscopy Showing Significant Monoatomic Ion Gradient Across The NE

Hernández-Cruz et al., 1990; Al-Mohanna et al., 1994; Himpens et al., 1994a, 1994b;
Gerasimenko et al., 1995, 1996; Bkaily et al., 1996; Kono et al., 1996; Tanaka et al., 1996; Lui et al., 1998a, 1998b; Pauly et al., 2000; Genka et al., 2000; Abrenica & Gilchrist, 2000

Luminescence Microscopy Showing Neglibible Monoatomic Ion Gradient Across The NE

Brini et al., 1994, 1996

Luminescence Microscopy Showing Significant Monoatomic Ion Gradient Across The NE

Badminton et al., 1995, 1996, 1998

The NPC Plug, ATP and NE Cisternal Ca²⁺

Greber & Gerace, 1995; Perez-Terzic et al., 1996, 1997;
Oberleithner, 1999; Rakowska et al., 1998; Strübing & Clapham, 1999; Stoffler et al., 1999 (press here to access their paper in PDF format); Perez-Terzic et al., 1999; Keminer & Peters, 1999; Wang & Clapham, 1999; Bustamante et al., 1999; Ribbeck & Gorlich, 2001; Shahin et al., 2001; Moore-Nichols et al., 2002;

Blockade of Ion Channel Activity by The NPC Antibody mAb414

Bustamante et al., 1995a; Prat & Cantiello, 1996

ER Contamination, Peripheral Channels and Other Vexing Issues

These and other issues are now officially discussed in our recent chapter of the Biophysical Society Textbook On-Line Bustamante, 2002c

IP₃- and Ryanodine-Sensitive Channels

Mak & Foskett, 1994; Stehno-Bittel et al., 1995a; Guihard et al., 1997; Mak et al., 1999, 2001a, 2001b;
Boehning et al., 2001a, 2001b; Moore-Nichols et al., 2002

Note 1: IP₃-receptors were found only on the inner membrane of the NE (e.g., Humbert et al., 1996).

Note 2: Heparin, used to identify nuclear IP₃-receptor operated channels, stabilizes the NPC plug (e.g., Strambio-de-Castillia et al., 1995), solubilizes chromatin (e.g., Bornens, 1973, 1977) and does alter other channels (e.g., Knaus et al., 1990; Lacinova et al., 1993; Hall et al., 1996; Krasilnikov et al., 1999; Sinnarajah et al., 1999). For a recent review on Nuclear Signaling, see Irvine, 2000 .

NE Ion Channels That May Be NPCs or May Be Related to NPCs Structurally or Functionally

Matzke et al., 1990, 1992; Mazzanti et al., 1990, 1991, 1994;
Innocenti & Mazzanti, 1993; Tabares et al., 1991; Maruyama et al., 1995;
Rousseau et al., 1996; Draguhn et al., 1997; Longin et al., 1997; Tonini et al., 1999; Schafer et al., 2002

NE Ion Channels Claimed To Be Not Relate to NPCs

Valenzuela et al., 1997, 2000; Grygorczyk & Grygorczyk, 1998;
Tonini et al., 2000; Franco-Obregon et al., 2000; Guihard et al., 2000

Patch-Clamp Measurement of NPC Diameter

Bustamante et al., 2000

Electrical Measurement of Non-NPC Ion Channels Diameter

Cecchi et al., 1982; Krasilnikov et al., 1992; 1995; 1996; 1998a; 1998b; Sabirov et al., 1992; Vodyanoy & Bezrukov, 1992; Carneiro et al., 1997; Cruickshank et al., 1997; Ternovsky et al., 1997; Merzlyak et al., 1999

Myosin and Actin as Key Mechanisms for NPC Gating

Tonini et al., 1999 have developed the idea that the NPC gate is determined by myosin and actin and that they are directly regulated by ATP and Ca²⁺. We disagree with this concept and maintain that ATP and Ca²⁺ act indirectly, through the NE cisterna and that macromolecular translocation explains and unifies all the observations ( Bustamante, 1994b; Bustamante et al., 1994c; Bustamante et al., 1995a; Bustamante & Varanda, 1998; Bustamante et al., 1999).

Microelectrodes Studies of The NE

Loewenstein & Kanno, 1962 (Nature 195:462), 1963 (J Cell Biol 16:421, J Gen Physiol 46:1123);
Kanno & Loewenstein, 1963 (Exp Cell Res 31:149);
Ito & Loewenstein, 1965; Kanno et al., 1965; Wiener et al., 1966;

Kislov & Veprintsev, 1970, 1971; Turkevich, 1970; Starodubov & Kurella, 1972;

Giulian & Diacumakos, 1977; Reynolds & Tedeschi, 1984

NPC Structure

Hinshaw et al., 1992; Akey & Radermacher, 1993; Panté & Aebi, 1996; Kiseleva et al., 1998; Danker & Oberleithner, 2000; Oberleithner et al., 2001 - (Full Text)

Nucleic Acid Translocation Silences Ion Channel Activity

Kasianowicz et al., 1996; Szabo et al., 1997, 1998; Hanss et al., 1998; Akeson et al., 1999; Lubensky & Nelson, 1999; Meller et al., 1999

Nuclear Pore Ion Channel Activity In Live Syncytial Nuclei

Bustamante, 2002b - see Additional Information

Nuclear Pore Genomics and Proteomics

Adam, 2001; Adam, 2001; Cronshaw et al., 2002, (full paper)

Nuclear Pore Reviews Relevant to Nuclear Electrophysiology

Prior to 2001: Bustamante, 1994b; Bustamante et al., 1994c; Matzke & Matzke, 1996; Stehno-Bittel et al., 1996; Perez-Terzic et al., 1997; Bustamante & Varanda, 1998 (HTML or PDF Reprint); Lee et al., 1998; Malviya & Rogue, 1998; Mazzanti, 1998; Petersen et al., 1998; Verkhratsky & Petersen, 1998 ; Stoffler et al., 1999; Rogue & Malviya, 1999 (full text); Bezrukov, 2000; Danker & Oberleithner, 2000; Kiseleva et al., 2000;
Beginning 2001: Mazzanti, Bustamante & Oberleithner, 2001 (see front cover below), Bustamante, 2000a

You may look at the paper by clicking the Front Cover above.
Front cover and paper link obtained with permission from the American Physiological Society.

Nuclear Electrophysiology Chapter in the Biophysical Society Textbook On-Line

Bustamante, 2002c

Press This Link for PubMed NPC Refs in Past 30 Days - Gateway to Further Bibliographic Search

Press This Link for PubMed NPC Reviews in Past 5 Years

We get most of our fluorescence probes from

NPC By AFM Images on right by Oberleithner and Krohne. Check also Amato, 1997a , b

Our Publications In Nuclear Physiology

2002 2001a, b 2000a, b 1998 1996 1995a, b, c 1994a, b, c 1993a 1992a

Get Some of The Latest Information on Signal Transduction

View or Get Promega's Signal Transduction Resource Guide

Attend On- or Off-Line NIH Videocast Conferences

Günter Blobel's Lecture (March 1st, 2000): Traffic Into and Out of The Nucleus

Nobel Laureates
Nobel Prizes in Physiology and Medicine (Lectures, Symposia, etc.)
Click on the links above for the full lists.
Click on the link below for a selected list.
Selected Nobel Prize Lectures, Symposia, etc.

Marine Biological Lab Conferences

GENOMICS

Human Genome

NIH Video-Symposium: Insights from the DNA Sequence of the Human Genome (February 12, 2001).
With Words From Watson And Crick

NIH Video-Conference: First Analysis of Complete Human Genome Press (February 12, 20001)

Nature Genomics Gateway

Front cover reprinted by permission from Nature 409: Feb 15, 2001 copyright 2001 Macmillan Magazines Ltd.

Science's Feb 16, 2001

First Plant Genome

Arabidosis Genome Slide Show (Nature 408, December 14, 2000)

Sites (contact us if we missed yours)

Transport Mechanisms from Dani Stoffler's Site

List of Scientists Working in This and Related Fields

Schematics Of Patch-Clamp Setup

Check out the Axon Instruments Patch-Clamp Instrumentation and Theory (The Axon Guide).

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