4.3.1 Dynamic Plasma Membrane Tether Force
A typical dynamic membrane tether force plot exhibits three different regions with respect to time (Fig.4.1): (1) tether formation, which starts from the onset of pulling, reaching the maximum tether force (Fmax), followed by a sudden reduction in force; (2) tether elongation, which begins after the reduction inFmax and continues until the desired tether length is reached, and (3) tether force relaxation, which starts once the tether elongation is halted, and continues until the force reaches an equilibrium value (Feq). The force profile shown in Fig.4.1was obtained from a control HEK cell. In this figure, Fmaxwas126 pN, and then the tether force dropped to61 pN. At the end of the tether elongation, the tether force reached a value ofFl71 pN. Finally, at the end of the relaxation, theFeqwas32 pN.
4.3.2 Plasma Membrane Tether Formation Force (F
max)
Tether formation force (Fmax) is defined as the force required to bend the plasma membrane, and separate it from the underlying cytoskeleton. The sudden force drop after reaching the maximum force value indicates the separation of the plasma membrane from cytoskeleton. Values of Fmax increased by depletion of membrane cholesterol content, and decreased as membrane was enriched with cholesterol. Specifically, meanstandard deviation (s.d.) values ofFmaxfor control cells were 13040 pN (n¼16). The respective membrane cholesterol concentration for control cells is 7.50.8 pmol/mg of protein. Means.d. values ofFmaxincreased to 17745 (n¼13) and 21845 pN (n¼7) when cells were incubated in DMEM containing 3 and 5 mM MbCD for cholesterol depletion, respectively. The respective membrane cholesterol concentrations are 6.60.3 and 5.7 0.8 pmol/mg of protein when cells were incubated in DMEM containing 3 and 5 mM MbCD. When cells were incubated in DMEM containing 3 and 5 mM cholesterol-MbCD to enrich the membrane cholesterol content of the cells, the means.d. values ofFmaxdecreased to 11030 (n¼7) and 9729 pN (n¼13), respectively. The respective membrane cholesterol concentrations are 14.51.9 and 17.30.6 pmol/mg of protein for cells incubated in 3 and 5 mM cholesterol-MbCD. Based on unpaired t-test analysis, the mean value ofFmaxfor control cells was significantly different from cholesterol-depleted cells, and the cells incubated in DMEM containing 5 mM cholesterol-MbCD for cholesterol enrichment (P<0.05).
Disruption of F-actin resulted in a significant decrease inFmaxin control cells from 13040 to 7227 pN (n¼13), indicating the significant role of cytoskeleton on the force required to bend and separate plasma membrane from the cytoskeleton, regardless of membrane composition manipulation. This is consistent with the observation of the decreased Fig. 4.1 Typical temporal
plasma membrane tether force plot for a HEK cell in response to a single-speed pulling protocol. Force plot shows tether formation (TF), tether elongation (TE), and tether force relaxation (REL) regions. Tether is formed and elongated at a constant pulling rate of 1mm/s to 20mm.Fmax- tether formation force,Feq- equilibrium tether force, F1-tether force at the end of elongation
26 N. Khatibzadeh et al.
Fmaxin cholesterol depleted and cholesterol enriched HEK cells upon F-actin disruption. Specifically, theFmaxdecreased to 8528 pN (n ¼7) in cells incubated in DMEM containing 5 mM MbCD for cholesterol depletion (P<0.05), and to 5528 pN (n¼7) in the cells incubated in DMEM containing 5 mM MbCD-cholesterol for cholesterol enrichment (P<0.05).
Comparing Fmaxamong the cells with intact F-actin, the significant increase inFmaxby cholesterol depletion suggests more membrane-cytoskeleton adhesion under cholesterol depleted conditions. Similarly, decreased values ofFmaxunder cholesterol enriched conditions suggest the weakened membrane-cytoskeleton adhesion.
4.3.3 Tether Formation Energy (E
tf)
The tether formation energy (Etf) is the work required to form the membrane tether, including the energy spent to bend the plasma membrane, viscous dissipations during tether formation, breakage of the plasma membrane-cytoskeleton bonds and separation of the membrane from the cytoskeleton, and any kind of plastic deformations of the composite plasma membrane- cytoskeleton during the tether formation process (TF region in Fig.4.1).
Optical tweezers combined with fluorescent microscopy provides a method to calculate the energy required to form the plasma membrane tethers based on the measurement of the temporal length of the locally deformed part of the cell bound to the bead. To calculate theEtf, the tether formation region (TF region in Fig. 4.1) of the dynamic tether force plots are examined and the time resolved displacement of the locally deformed part of the cell bound to bead (Xld) is calculated using the displacement of the PZT (XPZT) and the displacement of the trapped bead from the trapping center (Xbead) (Eq.4.1):
XldðtÞ ¼XPZTðtÞ XbeadðtÞ (4.1) Figure4.2aillustratesXld,XPZT, andXbeadin a bead-cell assembly where the cell is initially in contact with the bead, and when the cell is moving away which results in the application of a tangential force to the bead and therefore displacement of the trapped bead from the trapping centre. An example force–displacement profile in the tether formation region of the dynamic tether force profile is shown in Fig.4.2b, showing an initial linear rise followed by a nonlinear one.
The tether formation energy is calculated as the area under the force–displacement profiles (Fig. 4.2b) during tether formation region (TF region in Fig. 4.1). The meanstandard deviation (SD) of Etf for intact F-actin and F-actin Fig. 4.2 (a) Calculation of the local cell deformation length (Xld).Dashed linesshow initial cell-bead contact (no external force) and thesolid linesshow the bead-cell assembly while the cell is moving away to form membrane tether. (b) Force versus deformation of the tether formation region (TF) of the force plot shown in Fig.4.1
4 Effects of Membrane Composition and Cytoskeletal Proteins on Membrane Mechanics 27
disrupted HEK cells under cholesterol enriched and depleted conditions are shown in Fig.4.3. For control cells, theEtfis calculated101 40 (1018J). Incubation of the cells in DMEM containing 5 mM MbCD for cholesterol depletion resulted in significant increases ofEtfto 17950 (1018J). The energy of tether formation significantly decreases to 5825 (1018J) in response to cholesterol enrichment of the cells by incubating them in DMEM containing 5 mM cholesterol-MbCD.
Disruption of F-actin with Latranulin A caused significant decrease in tether formation energy in untreated cells and cholesterol depleted cells. Specifically,Etfdecreased to 5025 (1018J) and 4120 (1018J) in F-actin disrupted control and F-actin disrupted cholesterol depleted cells, respectively.
4.3.4 Plasma Membrane Tether Equilibrium Forces (F
eq)
When a membrane tether is elongated, the stretched membrane induces lateral tension within the cell body plasma membrane that draws more membrane and causes membrane flow into the tether. The membrane flow into the tether continues throughout the force relaxation region (REL region in Fig.4.1) until reaching an equilibrium value (Feq). The tether equilibrium force is a measure of the tension in the plasma membrane tether balanced against that within the cell body, and represents a zero net flow of membrane components into the tether.
The membrane composition significantly affected theFeqin HEK cells in our experiments. Specifically, mean value of Feqincreased in response to lowering the membrane cholesterol content, and decreased upon elevation of the membrane cholesterol in cells with intact F-actin. The means.d. values ofFeqfor control HEK cells were 3912 pN (n¼11).
This value significantly increased to 6616 (n¼10) and 7417 (n¼11) (P<0.05) when cells were incubated in DMEM containing 3 and 5 mM MbCD for cholesterol depletion, respectively. In response to incubating the cells with intact F-actin in DMEM containing 3 and 5 mM cholesterol-MbCD for cholesterol enrichment,Feqsignificantly decreased to 258 (n¼8) and 19.55 (n¼6), respectively (P<0.05).
In addition to the significant role of the membrane composition on theFeqvalue, the cytoskeletal F-actin integrity also significantly affected theFeqin our experiments. TheFeqsignificantly decrease in control cell upon F-actin disruption to 247 pN (n¼13). Given less plasma membrane-cytoskeleton association in F-actin disrupted cell, the significant decrease ofFeq suggests the prominent role of membrane-cytoskeleton associations in Feq; i.e. the tighter adhesions is associated with higherFeq, and weakened membrane-cytoskeleton adhesions is associated with lower values ofFeq.
Among the cells with intact F-actin, comparing theFeqvalues of the control cells and the cholesterol manipulated cells, the higherFeqvalues in cholesterol depleted cells suggest more adhesion of the membrane to the underlying cytoskeleton and similarly, lowerFeqvalues upon enrichment suggests less adhesion in response to cholesterol enrichment.
Fig. 4.3 Membrane tether formation energy versus plasma membrane cholesterol concentration for HEK cells with intact and disrupted F-actin
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