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Modeling Biophysical Mechanisms underlying Cellular Homeostasis
KTH, School of Engineering Sciences (SCI), Applied Physics, Cell Physics.
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cellular homeostasis is the effort of all living cells to maintain their intracellular content when facing physiological change(s) in the extracellular environment. To date, cellular homeostasis is known to be regulated mainly by time-consuming active mechanisms and via multiple signaling pathways within the cells. The aim of this thesis is to show that time-efficient passive (physical) mechanisms also, under the control and regulation of bio-physical factors such as cell morphology and distribution and co-localization of transport proteins in the cell membrane, can regulate cellular homeostasis. This thesis has been developed in an interface between physics and biology and focuses on critical cases in which cells face physiologically unstable environments at their steady state and therefore may need a constituent effort to maintain their homeostasis. The main hypothesis here is that the cell geometry is oriented in such a way that cellular homeostasis is preserved in a given environment. For exploring these cases, comparative spatial models have been developed that combine transporting function of membrane proteins with simple versus complex geometries of cells. Models confirm the hypothesis and show that cell morphology, size of extracellular space and intercellular distances are important for a dynamic regulation of water and ion homeostasis at steady state. The main clue is the existence of diffusion limited space (DLS) in the bulk extracellular space (ECS). DLS can, despite being ECS, maintain its ionic content and water balance due a controlled function of transport proteins in the membrane facing part of DLS. This can significantly regulate cellular water and ion homeostasis and play an important role in cell physiology. In paper I, the role of DLS is explored in the kidney whereas paper II addresses the brain.

The response of cells to change in osmolarity is of critical importance for water homeostasis. Cells primarily respond to osmotic challenge by transport of water via their membranes. As water moves into or out of cells, the volumes of intra- and extracellular compartments consequently change. Water transport across the cell membrane is enhanced by a family of water channel proteins (aquaporins) which play important roles in regulation of both cell and the extracellular space dimensions. Paper III explores a role for aquaporins in renal K+ transport. Experimentally this role is suggested to be different from bulk water transport. In a geometrical model of a kidney principal cell with several DLS in the basolateral membrane, a biophysical role for DLS-aquaporins is suggested that also provides physiological relevance for this study. The biophysical function of water channels is then extensively explored in paper IV where the main focus has been the dynamics of the brain extracellular space following water transport. Both modeling and experimental data in this paper confirmed the importance of aquaporin-4 expressed in astrocytes for potassium kinetics in the brain extracellular space.

Finally, geometrically controlled transport mechanisms are studied on a molecular level, using silicon particles as a simplified model system for cell studies (paper V and VI). In paper V the role of electrostatic forces (around the nano-pores and in between the loaded material and the silicon surface) is studied with regard to transport processes.  In paper VI the roles of pore size and molecular weight of loaded material are studied. All together this thesis presents various modeling approaches that employ biophysical aspects of transport mechanisms combined with cell geometry to explain cell homeostasis and address cell physiology-based questions.   

Place, publisher, year, edition, pages
Stockholm: KTH , 2010. , xii, 60 p.
Series
Trita-FYS, ISSN 0280-316X ; 2010:01
National Category
Condensed Matter Physics
Identifiers
URN: urn:nbn:se:kth:diva-11880ISBN: 978-91-7415-546-4 (print)OAI: oai:DiVA.org:kth-11880DiVA: diva2:287247
Public defence
2010-02-04, FA32, AlbaNova University Center, Roslagstullsbacken 21, KTH, Stockholm, 13:00 (English)
Opponent
Supervisors
Note
QC20100727Available from: 2010-01-21 Created: 2010-01-18 Last updated: 2010-07-27Bibliographically approved
List of papers
1. Role of diffusion limited space on water and salt homeostasis
Open this publication in new window or tab >>Role of diffusion limited space on water and salt homeostasis
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(English)Manuscript (preprint) (Other academic)
National Category
Condensed Matter Physics
Identifiers
urn:nbn:se:kth:diva-11365 (URN)
Note
QC 20100726Available from: 2009-10-30 Created: 2009-10-30 Last updated: 2010-07-27Bibliographically approved
2. Diffusion limited space contributes to K+ siphoning by regulation of K+ and water homeostasis in astrocytes
Open this publication in new window or tab >>Diffusion limited space contributes to K+ siphoning by regulation of K+ and water homeostasis in astrocytes
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Diffusion Limited Space (DLS) is defined as a region where diffusion is limited by the geometry. Two examples of DLS in the brain are the neuronal synapse, and the narrow region between astrocyte endfeet and blood capillaries. In a series of geometrical models we show that DLS plays a role in regulation of water and K+ homeostasis in the brain by an indirect functional coupling of aquaporins (AQPs) and inward rectifying K+ (Kir) channels in a membrane microdomain.

1. Simulations in geometrical models of a synapse region show that following a step increase in synaptic [K+], both K+ and water are taken up by astrocytes via AQPs and Kir channels lining the synapse.  This uptake creates a transient depletion of water in the synapse region that, enhanced by the DLS, facilitates K+ uptake and an efficient clearance of excess K+ from the synapse.

2. Simulations in a geometrical model of astrocytes show that the DLS formed between astrocyte endfeet and blood capillaries, facilitate the siphoning of accumulated K+ into the extracellular space facing the blood capillaries. The DLS geometry creates an efficient coupling between AQPs and Kir channels.

3. Furthermore, the models show that a local coupling between water and K+ transport is important for the maintenance of membrane potential and the net K+ spatial buffering capacity in the astrocytes.

4. In the full geometrical model of K+ spatial buffering we show that the geometry of the extracellular space both in the synapse region and in the endfeet is an essential component for the cell volume regulation.

Our results suggest that for regulation of K+ and water homeostasis in astrocytes, not only the classical aspects of functional couplings between proteins, but also the geometry of the cell and the microdomains are important. Further, our results suggest a central role for AQPs in the astrocyte endfeet and identify their contribution to K+ siphoning.

Identifiers
urn:nbn:se:kth:diva-11872 (URN)
Note
QC20100727Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2010-07-27Bibliographically approved
3. A role for AQP4 in renal K+ transport
Open this publication in new window or tab >>A role for AQP4 in renal K+ transport
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The principal cells of the collecting duct carry out two major tasks: concentration of urine and regulation of K+ homeostasis. Two water channels, AQP3 and AQP4, are expressed in the principal cell basolateral membrane. We propose that AQP4 participates in the regulation of K+ transport in the principal cells. K+ enters the cell via Na+, K+-ATPase-mediated transport in the basolateral membrane. The presence of K+ channels in this membrane permits some K+ recirculation, considered important for maintenance of membrane potential. Here we show that AQP4, but not AQP3, assembles with both Na+, K+-ATPase and an inwardly rectifying K+ channel Kir7.1. We hypothesize that AQP4, Na+, K+-ATPase and Kir7.1 form a K+-transporting microdomain and that AQP4 serves to maintain a favorable concentration gradient for K+ efflux into the diffusion-limited space within the deep infoldings in principal cell basal membrane. The hypothesis is tested in a mathematical model. The model predicts that the impact of AQP-mediated water transport on K+ transport is more significant if AQP water permeability is sensitive to fluctuations in extracellular K+ concentration ([K+]e). We measured water permeability of AQP4 expressed in a renal epithelial cell line and found that it is upregulated when [K+]e is increased to 8 mM, and downregulated when [K+]e is decreased to 1 mM. Studies in an oocyte system indicate that AQP4 does not possess a voltage or K+ sensor. Finally, we show that the expression of AQP4 in rat renal medulla is, in contrast to the expression of AQP2 and AQP3, resistant to changes in K+ intake. Our experimental data, together with the mathematical model, support the concept that AQP4 is involved in principal cell K+ transport processes.

Identifiers
urn:nbn:se:kth:diva-11874 (URN)
Note
QC20100727Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2010-07-27Bibliographically approved
4. Extracellular Space dynamics contribute to Potassium kinetics during cortical spreading depression in Aquaporin-4 Deficient Mice
Open this publication in new window or tab >>Extracellular Space dynamics contribute to Potassium kinetics during cortical spreading depression in Aquaporin-4 Deficient Mice
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Glial water channel aquaporin-4 (AQP4) plays an important role in neuroexcitation phenotypes that are highly associated with potassium homeostasis in brain extracellular space (ECS).  The mechanism of how AQP4 modulate the neuroexcitation through potassium redistribution during the neuronal signal transduction remains unknown.  Cortical spreading depression (CSD) is a self-propagating wave of neuronal depolarization with increased extracellular potassium concentration ([K+]o),  astrocyte swelling and subsequent severe contraction of ECS which provide a model for the mechanism study.  Here we characterized the CSD induced by KCl application in wild type (WT) and AQP4 deficient mice (AQP4-/-) and found AQP4-/- mice had a significant decrease in the frequency (6.9 ± 0.3 vs. 10.2 ± 0.5 CSDs/hr; p < 0.01), as well as the propagation velocity of CSD (2.9 ± 0.1 vs. 3.7 ± 0.1 mm/min; p < 0.05).  During CSD, the dynamic changes of extracellular potassium were determined using K+-selective microelectrodes and the extracellular space (ECS) was measured by TMA+ method.  We found the rate of K+ release and clearance was significantly prolonged in the AQP4-/- mice (t1/2, 10.2 ± 1.8 sec and 43.2 ± 2.3sec) compared to their WT counterparts (t1/2, 7.4 ± 0.3 sec and 35.7 ± 1.0 sec), which were paralleled by a significantly delayed contraction and recovery of ECS to baseline in AQP4-/- mice.  There is no difference in baseline or peak [K+]o. Importantly, no alterations were found in the expression or localization of inwardly rectifying K+ channel Kir4.1, gap junction hemichannel connexin-43, and anchor protein alpha-syntrophin in AQP4-/- mice.  A computer geometrical modeling of potassium accumulation and clearance during CSD confirmed the major role of ECS dynamic change in modulation of potassium kinetics. These results implicated an essential role of AQP4 in dynamic changes of ECS during CSD, which may be a new mechanism underlying the potassium kinetics and neuroexcitation.

Identifiers
urn:nbn:se:kth:diva-11875 (URN)
Note
QC20100727Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2010-07-27Bibliographically approved
5. Release and molecular transport of cationic and anionic fluorescent molecules in mesoporous silica spheres.
Open this publication in new window or tab >>Release and molecular transport of cationic and anionic fluorescent molecules in mesoporous silica spheres.
2008 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 24, no 19, 11096-102 p.Article in journal (Refereed) Published
Abstract [en]

We describe here a method for study of bulk release and local molecular transport within mesoporous silica spheres. We have analyzed the loading and release of charged fluorescent dyes from monodisperse mesoporous silica (MMS) spheres with an average pore size of 2.7 nm. Two different fluorescent dyes, one cationic and one anionic, have been loaded into the negatively charged porous material and both the bulk release and the local molecular transport within the MMS spheres have been quantified by confocal laser scanning microscopy. Analysis of the time-dependent release and the concentration profiles of the anionic dye within the spheres show that the spheres are homogeneous and that the release of this nonadsorbing dye follows a simple diffusion-driven process. The concentration of the cationic dye varies radially within the MMS spheres after loading; there is a significantly higher concentration of the dye close to the surface of the spheres (forming a "skin") compared to that at the core. The release of the cationic dye is controlled by diffusion after an initial period of rapid release. The transport of the cationic dye within the MMS spheres of the dye from the core to near the surface is significantly faster compared to the transport within the surface "skin". A significant fraction of the cationic dye remains permanently attached to the negatively charged walls of the MMS spheres, preferentially near the surface of the spheres. Relating bulk release to the local molecular transport within the porous materials provides an important step toward the design of new concepts in controlled drug delivery and chromatography.

Identifiers
urn:nbn:se:kth:diva-11877 (URN)10.1021/la801179v (DOI)000259673500084 ()18767822 (PubMedID)2-s2.0-54549109154 (Scopus ID)
Note
QC20100727Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2010-07-27Bibliographically approved
6. Intraparticle transport and release of dextran in silica spheres with cylindrical mesopores
Open this publication in new window or tab >>Intraparticle transport and release of dextran in silica spheres with cylindrical mesopores
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2010 (English)In: Langmuir, ISSN 0743-7463, E-ISSN 1520-5827, Vol. 26, no 1, 466-70 p.Article in journal (Refereed) Published
Abstract [en]

The transport of oligomeric molecules in silica spheres with cylindrical mesopores has been quantified and related to the structural features of the spherical particles and the interactions at the solid-liquid interface. An emulsion-solvent evaporation method was used to produce silica spheres having cylindrical mesopores with an average pore diameter of 6.5 nm. The transport of dextran molecules (fluorescently tagged) with molecular weights of 3000 and 10,000 g/mol was quantified using confocal laser scanning microscopy (CLSM). The intraparticle concentration profiles in the dextran-containing spheres were flat at all times, suggesting that the release is not isotropic and not limited by diffusion. The release of dextran into the solution is characterized by an initial burst, followed by long-term sustained release. The release follows a logarithmic time dependency, which was rationalized by coupling concentration-dependent effective diffusion constants with adsorption/desorption.

Keyword
MICELLE-TEMPLATED SILICA, ADSORPTION, DIFFUSION, GAS, MICROSCOPY, PARTICLES, MOLECULES, KINETICS, SORPTION, ALUMINA
National Category
Chemical Sciences
Identifiers
urn:nbn:se:kth:diva-11878 (URN)10.1021/la902092e (DOI)000272937500062 ()19769352 (PubMedID)2-s2.0-73649097538 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20100727Available from: 2010-01-18 Created: 2010-01-18 Last updated: 2011-01-25Bibliographically approved

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