Presenting antigen presentation in living cells using biophysical techniques

Caveolae are small cell surface invaginations, important for control of membrane tension, signaling cascades and lipid sorting. Their formation is coupled to the lipid-dependent oligomerization of the proteins Caveolin1 and Cavin1, which are essential for membrane curvature generation. Yet, the mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical techniques to show that Cavin1 inserts into membranes. We found that the helical region 1 (HR1) initiated membrane binding via electrostatic interactions, which is further enforced by partial helical insertion in a PI(4,5)P2-dependent process mediated by the disordered region 1 (DR1). In agreement with this, the DR1 was found important for the co-assembly of Cavin1 with Caveolin1 in living cells. We propose that DR1 and HR1 of Cavin1 constitute a novel membrane interacting unit facilitating dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

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Visualizing the action of steroid hormone receptors in living cells

Nuclear Receptor Signaling ◽

10.1621/nrs.05003 ◽

2007 ◽

Vol 5 (1) ◽

pp. nrs.05003 ◽

Cited By ~ 42

Author(s):

Alexander Griekspoor ◽

Wilbert Zwart ◽

Jacques Neefjes ◽

Rob Michalides

Keyword(s):

Scientific Community ◽

Hormone Receptors ◽

Steroid Hormone ◽

Living Cells ◽

Steroid Hormone Receptors ◽

Complex Nature ◽

Physiological Processes ◽

Biophysical Techniques ◽

Native Context ◽

External Stimuli

Transcription controlled by Steroid Hormone Receptors (SHRs) plays a key role in many important physiological processes like organ development, metabolite homeostasis, and response to external stimuli. Understandably, the members of this family have drawn a lot of attention from the scientific community since their discovery, four decades ago. Still, after many years of research we are only beginning to unravel the complex nature of these receptors. The pace at which we do has improved significantly in recent years with the discovery of genetically encoded fluorescent probes, and the accompanying revival of biophysical approaches that allow more detailed study of SHRs. Here, we will look into the different aspects of SHR signalling, and discuss how biophysical techniques have contributed to visualizing their function in their native context, the living cell.

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Biophysical Techniques for Detection of cAMP and cGMP in Living Cells

International Journal of Molecular Sciences ◽

10.3390/ijms14048025 ◽

2013 ◽

Vol 14 (4) ◽

pp. 8025-8046 ◽

Cited By ~ 54

Author(s):

Julia Sprenger ◽

Viacheslav Nikolaev

Keyword(s):

Living Cells ◽

Biophysical Techniques ◽

Camp And Cgmp

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Fluorescence ratio imaging of dynamic intracellular ionic signals

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102298 ◽

1988 ◽

Vol 46 ◽

pp. 42-43

Author(s):

R. Y. Tsien ◽

A. Minta ◽

M. Poenie ◽

J.P.Y. Kao ◽

A. Harootunian

Keyword(s):

Binding Sites ◽

Living Cells ◽

Molecular Engineering ◽

Ph Indicators ◽

Highly Sensitive ◽

Fluorescence Ratio Imaging ◽

Ratio Imaging ◽

Technical Advances ◽

Indicator Dyes ◽

Fluorescein Dyes

Recent technical advances now enable the continuous imaging of important ionic signals inside individual living cells with micron spatial resolution and subsecond time resolution. This methodology relies on the molecular engineering of indicator dyes whose fluorescence is strong and highly sensitive to ions such as Ca2+, H+, or Na+, or Mg2+. The Ca2+ indicators, exemplified by fura-2 and indo-1, derive their high affinity (Kd near 200 nM) and selectivity for Ca2+ to a versatile tetracarboxylate binding site3 modeled on and isosteric with the well known chelator EGTA. The most commonly used pH indicators are fluorescein dyes (such as BCECF) modified to adjust their pKa's and improve their retention inside cells. Na+ indicators are crown ethers with cavity sizes chosen to select Na+ over K+: Mg2+ indicators use tricarboxylate binding sites truncated from those of the Ca2+ chelators, resulting in a more compact arrangement of carboxylates to suit the smaller ion.

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Application of FRAP and digitized fluorescence microscopy to problems in cell membrane dynamics

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100102675 ◽

1988 ◽

Vol 46 ◽

pp. 118-119

Author(s):

K. Jacobson ◽

A. Ishihara ◽

B. Holifield ◽

F. Zhang

Keyword(s):

Fluorescence Microscopy ◽

Cell Biology ◽

Extended Period ◽

Dynamic Structure ◽

Living Cells ◽

Membrane Dynamics ◽

Molecular Cell Biology ◽

Image Averaging ◽

Single Living Cells ◽

Single Living

Our laboratory is concerned with understanding the dynamic structure of the plasma membrane with particular reference to the movement of membrane constituents during cell locomotion. In addition to the standard tools of molecular cell biology, we employ both fluorescence recovery after photo- bleaching (FRAP) and digitized fluorescence microscopy (DFM) to investigate individual cells. FRAP allows the measurement of translational mobility of membrane and cytoplasmic molecules in small regions of single, living cells. DFM is really a new form of light microscopy in that the distribution of individual classes of ions, molecules, and macromolecules can be followed in single, living cells. By employing fluorescent antibodies to defined antigens or fluorescent analogs of cellular constituents as well as ultrasensitive, electronic image detectors and video image averaging to improve signal to noise, fluorescent images of living cells can be acquired over an extended period without significant fading and loss of cell viability.

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Multimode light microscopy and fluorescence-based reagents as tools for the study of chemical and molecular dynamics of living cells and tissues

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100122496 ◽

1992 ◽

Vol 50 (1) ◽

pp. 418-419

Author(s):

D. L. Taylor

Keyword(s):

Living Cells ◽

Cellular Level ◽

Molecular Techniques ◽

Cellular Functions ◽

Macromolecular Assemblies ◽

Cell Functions ◽

Molecular Events ◽

Yield Information ◽

Full Knowledge ◽

Structural Methods

Cells function through the complex temporal and spatial interplay of ions, metabolites, macromolecules and macromolecular assemblies. Biochemical approaches allow the investigator to define the components and the solution chemical reactions that might be involved in cellular functions. Static structural methods can yield information concerning the 2- and 3-D organization of known and unknown cellular constituents. Genetic and molecular techniques are powerful approaches that can alter specific functions through the manipulation of gene products and thus identify necessary components and sequences of molecular events. However, full knowledge of the mechanism of particular cell functions will require direct measurement of the interplay of cellular constituents. Therefore, there has been a need to develop methods that can yield chemical and molecular information in time and space in living cells, while allowing the integration of information from biochemical, molecular and genetic approaches at the cellular level.

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