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Dr. Sharpless visits Dr. Jena's Lab

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Dr. K. Barry Sharpless (2001 Nobel Laureate in Chemistry) with a few members of the Jena Lab, during his visit on 9/22/15.

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of Dynamics of Cellular Secretion)

Symposium 2002

Manuscripts with Cover 2002


Manuscripts with Cover 2009


Manuscripts with Cover 2010

Manuscripts with Cover 2012

Manuscripts with Cover 2013


Books Published



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Topology of the apical cell surface of isolated pancreatic acini, observed using AFM.  Scattered 'pits' at the apical plasma membrane are seen.  One 'pit' (Inset) with four 'depressions' is shown. A number of large pore-like structures are also identified [Proc. Natl. Acad. Sci. USA 1997. 94:316-321].

Dynamics of depressions following stimulation of secretion. A. Several depressions within a pit are shown.  The scan line across three depressions in the top panel is represented graphically in the middle panel and defines the diameter and relative depth of the depressions; the middle depression is represented by red arrowheads.  The bottom panel represents percent of total cellular amylase release in the presence and absence of the secretagogue Mas 7.  B. Notice an increase in the diameter and depth of depressions, correlating with an increase in total cellular amylase release at 5 min after stimulation of secretion.  C. At 30 min after stimulation of secretion, there is a decrease in diameter and depth of depressions, with no further increase in amylase release over the 5 min time point.  No significant increase in amylase secretion or depression diameter were observed in resting acini or those exposed to the nonstimulatory mastoparan analog Mas 17 [Proc. Natl. Acad. Sci. USA 1997. 94:316-321].

AFM micrographs of fixed cells demonstrating amylase-specific immunogold localization at 'pits' and 'depressions'.  Following stimulation of secretion, the acinar cell was exposed to amylase-specific antibody followed by 30 nm gold-protein A, and fixed (a).  Note gold decorating 'pit' and depressions at the apical end of the cell (b) [yellow arrowheads].  A high magnification of one 'depression' is shown in (c), with gold decorating its edges.  Similar to live acinar cells, little morphology is seen at the basolateral end of the cell (d), and no gold is localized at this end.  Several AFM micrographs of 'pits' and the 'depressions' from a number of experiments depict the specific immunolocalization of amylase-specific immunogold (yellow spots; e-m) [Cell Biology International. Jan. 2002, 26 (1):35-42].

Schematic diagram depicting 'pits' and 'depression' at the cell plasma membrane, where secretory vesicles dock and transiently fuse to release vesicular contents [Cell Biology International. Jan. 2002, 26 (1):35-42].

AFM and immunoAFM micrographs of the fusion pore, demonstrating pore morphology and the release of secretory products at the site. (a). A pit with four fusion pores within, found at the apical surface in a live pancreatic acinar cell.  (b). Following stimulation of secretion, amylase-specific immunogold localize at the pit and fusion pores within, demonstrating them to be secretory release sites.  (c).  Some fusion pores demonstrate greater immunogold localization, suggesting more release of amylase from them.  (d). AFM micrograph of a single fusion pore in a live acinar cell [Biophysical J. 2003, 84: 1337-1343].

Morphology of the cytosolic side of the fusion pore revealed in AFM studies on isolated pancreatic plasma membrane preparations.  (a). AFM micrograph of isolated plasma membrane preparation reveals the cytosolic end of a pit with inverted cup-shaped structures, the fusion pores.  Note the 600nm in diameter ZG at the left hand corner of the pit.  (b). Higher magnification of the same pit showing clearly the 4-5 fusion pores within.  (c). The cytosolic end of a single fusion pore is depicted in this AFM micrograph.  (d). Immunoblot analysis of 10mg and 20mg of pancreatic plasma membrane preparations, using SNAP-23 antibody, demonstrates a single 23kDa immunoreactive band.  (e,f).  The cytosolic side of the plasma membrane demonstrating the presence of a pit with a number of fusion pores within, shown prior to (e) and following addition of the SNAP-23 antibody (f).  Note the increase in height of the fusion pore cone base revealed by section analysis (bottom pannel), demonstrating localization of SNAP-23 antibody to the base of the fusion pore [Biophysical J. 2003, 84: 1337-1343].

Transmission Electron Micrograph of the fusion pore showing vertical and lateral structures, giving it a basket-like appearance.  (a). Two fusion pores at the apical plasma membrane of a pancreatic acinar cell, with a docked zymogen granule.  (b). High resolution image of one of the fusion pores clearly shows three lateral and a number of vertical ridges, giving it a basket-like appearance.  (c). An isolated zymogen granule associated with a fusion pore, reveals (d). clearly the lateral and vertical structures of the fusion pore complex.  Scale = 100nm [Biophysical J. 2003, 84: 1337-1343].

The fusion pore complex.  (a). Electron micrograph of a fusion pore, with positions of the vertical and lateral structures depicted by red and yellow dashed lines for clarity.  (b). A schematic model of the fusion pore showing (c) the presence of t-SNAREs at the base of the basket, where the v-SNARE associated ZG can dock.  (d). Thus, the t- and v-SNAREs associated in apposing bilayers can interact in a circular array (yellow ring) to establish continuity between ZG contents and the fusion pore.  (e). When v-SNARE reconstituted artificial lipid vesicles are allowed to interact with a t-SNARE-reconstituted lipid support, the t-/v-SNAREs interact in a circular array as shown in the AFM micrograph on the right.  The pore formed by t-/v-SNARE interaction, is conducting, as demonstrated by electrophysiological measurements [Biophysical J. 2003, 84: 1337-1343].

AFM micrographs and force plots of mica and lipid surface and of SNAREs on lipid membrane.  AFM performed on freshly cleaved mica (A, left), and on lipid membrane formed on the same mica surface (A, right), demonstrating differences in the force vs. distant curves.  Note the curvilinear shape exhibited in the force vs. distant curves of the  lipid surface in contrast to mica.  Three dimensional AFM micrographs of neuronal t-SNAREs deposited on the lipid membrane (B), and following the addition of v-SNARE (C).  Section analysis of the SNARE complex in (B) and (C) is depicted in (D).  Note the smaller curve belonging to the t-SNARE complex in (B), is markedly enlarged following addition of v-SNARE.  Artificial bilayer lipid membranes are nonconducting either in the presence or absence of SNAREs (E, F).  Current verses time traces of bilayer membranes containing proteins involved in docking and fusion of synaptic vesicles while the membranes are held at 60 mV (current/reference voltage). (E) When t-SNAREs are added to the planar lipid bilayer containing the synaptic vesicle protein, VAMP-2, no occurrence of current spike for fusion event at the bilayer membrane is observed (n=7).  (F) Similarly, no current spike is observed when t-SNAREs (syntaxin 1A-1 and SNAP25) are added to the cis side of a bilayer chamber following with VAMP-2.   Increasing the concentration of t-SNAREs and VAMP-2 protein [Biophysical J. 2002, 83: 2522-2527].

Pore-like structures are formed when t-SNAREs and v-SNARE in opposing bilayers interact.  (A) Unfused v-SNARE vesicles on t-SNARE reconstituted lipid membrane. (B) Dislodgement and/or fusion of v-SNARE-reconstituted vesicles with a t-SNARE-reconstituted  lipid membrane, exhibit formation of pore-like structures due to the interaction of v- and t-SNAREs in a circular array. The size of the pores range between 50-150 nm (B-D).  Several 3D AFM amplitude images of SNAREs arranged in a circular array (C) and some at higher resolution (D), illustrating a pore-like structure at the center is depicted.  Scale bar is 100 nm.  Recombinant t-SNAREs and v-SNARE in opposing bilayers drive membrane fusion.  (E) When t-SNARE vesicles were exposed to v-SNARE reconstituted bilayers, vesicles fused.  Vesicles containing nystatin/ergosterol and t-SNAREs were added to the cis side of the bilayer chamber. 
Fusion of t-SNARE containing vesicles with the membrane observed as current spikes that collapse as the nystatin spreads into the bilayer membrane.  To determine membrane stability, the transmembrane gradient of KCl was increased, allowing gradient driven fusion of nystatin-associated vesicles [Biophysical J. 2002, 83: 2522-2527]

Opposing bilayers containing t- and v-SNAREs respectively, interact in a circular array to form conducting pores.  (A) Schematic diagram of the bilayer-electrophysiology setup. (B) Lipid vesicle containing nystatin channels (in red) and both vesicles and membrane bilayer without SNAREs, demonstrate no significant changes in capacitance and conductance. Initial increase in conductance and capacitance   may  be due to vesicle-membrane attachment.  To demonstrate membrane stability (both bilayer membrane and vesicles), the transmembrane gradient of KCl was increased to allow gradient driven fusion and a concomitance increase of conductance and capacitance.  (C) When t-SNARE vesicles were added to a v-SNARE membrane support, the SNAREs in opposing bilayers arranged in a ring pattern, forming pores (as seen in the AFM micrograph on the extreme right) and there were seen  stepwise increases in capacitance and conductance (-60 mV holding potential).  Docking and fusion of the vesicle at the bilayer membrane, opens vesicle-associated nystatin channels and SNARE-induced pore formation, allowing conductance of ions from cis to the trans side of the bilayer membrane.  Then further addition of KCl to induce gradient driven fusion, resulted in little or no further increase in conductance and capacitance, demonstrating docked vesicles have already fused [Biophysical J. 2002, 83: 2522-2527].

A hypothetical model illustrating the possible mechanism and cycle of synaptic vesicle fusion at the presynaptic membrane in neurons. Coiling and super-coiling of native neuronal SNAREs and long-rods, and their possible implication in membrane fusion is depicted. The v-SNARE (VAMP) on docked synaptic vesicles, and t-SNARE (syntaxin, SNAP-25) at the presynaptic membrane, form the SNARE complex. Following a secretory stimuli, the SNARE complex and the long-rod probably undergo coiling and super-coiling, resulting in juxtaposition of the two membranes and generation of centrifugal strain at the contact points between the two membranes. The close apposition of both membranes and the centrifugal strain (force) at their points of contact may result in lipid mixing and fusion of the two bilayers. Following fusion, and expulsion of vesicular contents, the complex is disassembled in the presence of NSF-ATP, in preparation for the next round of vesicle fusion. Right panel depicts the cover in that issue [Cell Biol. Int.  (1999), 22:657-670].

Isolated ZGs ranging in size from 0.2 to 1.2 m obtained from rat pancreas as seen by electron and AFM. (a) An electron micrograph of the electron-dense ZGs. Note the purity of the ZG preparation. (Bar = 1 m.) (b) A three-dimensional AFM image of isolated ZGs adhering to a Cell-Tak-coated mica sheet. Notice the size heterogeneity in the ZG population [Proc. Natl. Acad. Sci. 1997. 94:13317-13322].

mmunolocalization and biochemical detection of a Gi protein with ZGs from rat exocrine pancreas. (a and b) Immunoblot analysis. Identification of G alpha i3-immunoreactive antigen associated with ZGM. (a) Ten micrograms each of TH and ZGM was resolved using a single dimension 12.5% SDS gel electrophoresis before electrotransfer on to nitrocellulose and immunoblotting. A 44-kDa immunoreactive band was detected in both fractions but enriched in the ZGM. (b) Twenty five micrograms of ZGM protein was resolved on two-dimensional 16-BAC gel electrophoresis before electrotransfer and immunoblotting using the G alpha i3-specific antibody. Note a single spot at  approx 44 kDa, suggesting that only one G alpha i3 isoform is present in the ZGM. (c) Detection of Gi-specific GTPase activity associated with ZG. GTPase activity in 25 g of ZG incubated at 30C for 5 min in the presence of varying concentrations of either Mas7 or Mas17 demonstrates a dose-dependent increase in activity in the presence of Mas7, which is absent in the presence of Mas17. Values are mean  SE [Proc. Natl. Acad. Sci. 1997. 94:13317-13322]

Increase in size of ZGs in the presence of GTP. (a-c) Two-dimensional AFM images of the same granules after exposure to 20 M GTP at time 0 (a), 5 minutes (b), and 10 minutes (c). (d-f) The same granules are shown in three-dimensions: the three-dimensional image of the granules at time 0, 5 minutes, and 10 minutes, respectively, after exposure to GTP. (g-i) The GTP-induced increase in size of another group of ZGs observed by confocal microscopy. Confocal images of the same ZGs at time 0, 5 minutes, and 10 minutes after GTP exposure are shown. (Bar = 1 m.) Values represent one of three representative experiments.

AQP1 immunoreactivity is tightly associated with the ZG membrane fraction. Total homogenate (T), zymogen granule (Z), 200,000  g particulate (P), and supernatant (S) from total homogenates, and zymogen granule membrane (M), was resolved by using 12.5% SDS/PAGE. The 28-kDa AQP1 immunoreactivity is associated with the M fraction (A). The purity of isolated Z is demonstrated by the enriched presence of VAMP in the M fraction (B). ZGs are membrane-bound secretory vesicles packed with secretory proteins. (C). After treatment of the granule membrane, the particulate and supernatant fractions were separated by centrifugation at 200,000  g. Examination of particulate (C-P) and supernatant (C-S) fractions prepared after PBS treatment, treatment with 1% Triton X-100 in PBS (T-P, T-S), or with 1 M KCl (N-P, N-S) demonstrates specific and tight association of AQP1 immunoreactivity with M. Exposure of the M to PBS or 1 M KCl failed to dislodge the AQP1 immunoreactivity; however, the nonionic detergent Triton X-100 was able to dissociate AQP1 from the granule membrane [Proc. Natl. Acad. Sci. USA. 2002. 99(7):4720-4724].

Immunogold electron microscopic (10-nm particles) localization of AQP1 to ZG (arrowheads). Note some AQP1 associated with the cell plasma membrane (arrow). (Magnification: 57,000.) [Proc. Natl. Acad. Sci. USA. 2002. 99(7):4720-4724]. 

Sensitivity of GTP-induced ZG swelling. (A-H) Three-dimensional atomic force microscopy images and section analysis of ZG untreated (A and B) or pretreated with 25 M HgCl2 (C and D), pretreated with 25 M HgCl2 followed by 100 M 2-mercaptoethanol (E and F), before (A, C, and E) and after (B, D, and F) exposure to 40 M GTP. Notice the increase in size of ZG (from A to B and I) in the three-dimensional array, section analysis, and bar graph after exposure to GTP. Pretreatment of ZG with HgCl2 abolishes the GTP effect (from C to D and I). However, the inhibitory effect of HgCl2 is reversed partially in the presence of 2-mercaptoethanol (from E to F and I). Incubation of ZG in water instead of 125 mM KCl-Mes buffer, pH 6.5, has little effect on ZG size [time 0 min (G) to time 30 min (H and I).]

GTP-induced ZG swelling is rapid and involves HgCl2-sensitive 3H2O entry. (A and B) The height and width of a single ZG (yellow arrow) is monitored in seconds after exposure to 40 M GTP. Notice the linear time-dependent increase in both height and width of the ZG after exposure to GTP. (C) A GTPdose dependent increase in tritiated water entry is observed in ZG. (D) Exposure of ZG to HgCl2 inhibits both basal and GTP-induced tritiated water entry into ZG [Proc. Natl. Acad. Sci. USA. 2002. 99(7):4720-4724].

AQP1-specific antibody binds to the ZG membrane and blocks water traffic. (A) Immunoblot assay demonstrating the presence of AQP1 antibody in SLO-permeabilized ZG. Lanes: 1, AQP1 antibody alone; 2, nonpermeable ZG exposed to antibody; 3, permeable ZG exposed to AQP1 antibody. Immunoelectron micrographs of intact ZGs exposed to AQP1 antibody demonstrate little labeling (B and C). (Bar = 200 nm.) Contrarily, SLO-treated ZG demonstrate intense gold labeling at the luminal side of the ZG membrane (D and E). AQP1 regulates GTP-induced water entry in ZG. (F) Schematic diagram of ZG membrane depicting AQP1-specific antibody binding to the carboxyl domain of AQP1 at the intragranular side to block water gating. (G, H, and K) AQP1 antibody introduced into ZG blocks GTP-induced water entry and swelling (from G to H, after GTP exposure). (I-K) However, only vehicle introduced into ZG retains the GTP-stimulatable effect (from I to J, after GTP exposure) [Proc. Natl. Acad. Sci. USA. 2002. 99(7):4720-4724].

Schematic diagram depicting the role of ZGM-associated Gai3 in regulation of secretory vesicle swelling and expulsion of vesicular content. In the presence of GTP, the Gai3 protein is activated by replacement of its GDP with GTP, resulting in the opening of ZGM-associated Cl- , K+ and water channels. There is then a net increase in flow of Cl- and K+ ions together with water molecules into the ZG lumen, resulting in vesicle swelling and expulsion of vesicular contents.

Fusion Pore and Porosome: the secretory machinery at the cell plasma membrane.  Porosomes in the exocrine pancreas (top panels) and neurons (bottom panels).  In the top left panel, an electron micrograph of a single porosome at the apical plasma membrane (PM) of a pancreatic acinar cell is shown.  Note the porosome membrane (POM, yellow arrowhead) associated with the membrane of a secretory vesicle (ZGM).  A circular ring structure (blue arrowhead) forms the neck of the porosome complex.  On the top right panel, is an atomic force micrograph of the apical end of a live pancreatic acinar cell, demonstrating the presence of four porosomes (one pointed by the yellow arrowhead).  Porosomes in the exocrine pancreas range in size from 100 - 180 nm.  In the bottom left panel, an electron micrograph of a neuronal porosome (red arrowhead) in association with a synaptic vesicle (SV) at the presynaptic membrane (Pre-SM) of the nerve terminal, is demonstrated.  Note a central plug in the porosome complex.  The bottom right panel is an atomic force micrograph of a neuronal porosome at the presynaptic membrane in live cell.  Note the central plug (red arrowhead).  The neuronal porosome is an order of magnitude smaller (10-12 nm) than the porosome in the exocrine pancreas


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