6 research outputs found
Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells.
Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells
Biophysical investigations of cells focusing on the utility of optical tweezers : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Physics at Biophysics and Soft Matter Group, School of Fundamental Science, Massey University, New Zealand
Figures 1.1, 1.2, 1.3, 2.4, 2.5, 2.7, 4.3 (a, b & c), 4.4 & 4.5 are re-used with permission. Figures 1.5 and 6.1 are re-used under a Creative Commons Attribution 3.0 Unported (CC BY 3.0) license and Attribution 4.0 International (CC BY 4.0) license respectively.The aim of this thesis was to explore the utility of different biophysical techniques,
particularly optical tweezers (OT), in the investigation of the mechanical properties
and interactions of biological samples. Specifically, MCF7 cells and their extracted nuclei
were investigated mechanically, while the adhesion property of selected bacteria
to the milk fat globule was also used as an exemplar. Biological cells have the ability
to actively respond to external mechanical forces exerted by the microenvironment.
The cellular response can be viscous, elastic, or viscoelastic in nature depending on
the nature of the applied forces and the mechanical stresses applied. Changes in the
mechanical properties of cells and nuclei have emerged as a prominent hallmark of
many human diseases, particularly in neurodegenerative and metastatic diseases.
In this thesis, to understand the application of these techniques to biological systems
better, bulk rheology and microrheology studies were first performed on a model
viscoelatic fluid (PEO). Particularly, the passive and active microrheology of this model
viscoelastic material was characterized using optical tweezers and video particle tracking
to develop the prerequisite experimental and analytical methods.
Using the experimental knowledge gained from applying optical tweezers to standard
materials, a mechanistic approach was developed in order to better understand
how the mechanical properties of MCF7 cells change when the amount of heterochromatin
protein (HP1a) present inside the nuclei was reduced. (HP1a) is an architectural
protein that establishes and maintains heterochromatin, ensuring genome fidelity and
nuclear integrity. While the mechanical effects of changes in the relative amount of euchromatin
and heterochromatin brought about by inhibiting chromatin modifying enzymes
have been studied previously, here we measure how the material properties of
the cells are modified following the knockdown HP1a. Indentation experiments using
optical tweezers revealed that the knockdown cells have apparent Young’s modului
significantly lower than control cells. Similarly, tether experiments performed using
optical tweezers revealed that the membrane tensions of knockdown cells were lower
than those of control cells. This assay led to further work on studying the mechanical
properties of nuclei extracted from MCF7 cells.
A combination of atomic force microscopy, optical tweezers, and techniques based
on micropipette aspiration was used to characterize the mechanical properties of nuclei
extracted from HP1a knockdown or matched control cells. Similar to the previous
finding on cells, local indentation performed using atomic force microscopy and
optical tweezers found that the knockdown nuclei have apparent Young’s modului
significantly lower than control nuclei. In contrast, results from pipette-based techniques
in the spirit of microaspiration, where the whole nuclei were deformed and
aspirated into a conical pipette, showed considerably less variation between HP1a
knockdown and control, consistent with previous studies reporting that it is predominantly
the lamins in the nuclear envelope that determine the mechanical response
to large whole-cell deformations. The differences in chromatin organisation observed
by various microscopy techniques between the MCF7 control and HP1a knock-down
nuclei correlated well with the results of our measured mechanical responses and our
hypotheses regarding their origin.
Finally, not just the mechanical properties of the cells but also their interactions (an
interaction between the milk fat globule membrane and two bacterial strains - Lactobacillius
fermentum strains - 1487 and 1485) was explored as a side project by probing
with optical tweezers. The difference in bacterial cell surface properties of these two
strains and its effects on intestinal epithelial barrier integrity has already been studied.
This study focuses on measuring the adhesion force between membrane and bacteria
using optical tweezers. The results suggested that L. fermentus AGR1487 strongly interacts
with MFGM compared to AGR1485. All in all, this thesis demonstrates how
biophysical techniques can provide valuable insights into understanding biological
systems
Depletion of HP1α alters the mechanical properties of MCF7 nuclei
Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young’s moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin
Depletion of HP1α alters the mechanical properties of MCF7 nuclei
Within the nucleus of the eukaryotic cell, DNA is partitioned into domains of highly condensed, transcriptionally silent heterochromatin and less condensed, transcriptionally active euchromatin. Heterochromatin protein 1α (HP1α) is an architectural protein that establishes and maintains heterochromatin, ensuring genome fidelity and nuclear integrity. Although the mechanical effects of changes in the relative amount of euchromatin and heterochromatin brought about by inhibiting chromatin-modifying enzymes have been studied previously, here we measure how the material properties of the nuclei are modified after the knockdown of HP1α. These studies were inspired by the observation that poorly invasive MCF7 breast cancer cells become more invasive after knockdown of HP1α expression and that, indeed, in many solid tumors the loss of HP1α correlates with the onset of tumor cell invasion. Atomic force microscopy (AFM), optical tweezers (OT), and techniques based on micropipette aspiration (MA) were each used to characterize the mechanical properties of nuclei extracted from HP1α knockdown or matched control MCF7 cells. Using AFM or OT to locally indent nuclei, those extracted from MCF7 HP1α knockdown cells were found to have apparent Young's moduli that were significantly lower than nuclei from MCF7 control cells, consistent with previous studies that assert heterochromatin plays a major role in governing the mechanical response in such experiments. In contrast, results from pipette-based techniques in the spirit of MA, in which the whole nuclei were deformed and aspirated into a conical pipette, showed considerably less variation between HP1α knockdown and control, consistent with previous studies reporting that it is predominantly the lamins in the nuclear envelope that determine the mechanical response to large whole-cell deformations. The differences in chromatin organization observed by various microscopy techniques between the MCF7 control and HP1α knockdown nuclei correlate well with the results of our measured mechanical responses and our hypotheses regarding their origin
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Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells.
Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells
Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells