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1 The Cell Nanosized Components, Mechanics, and Diseases

1 The Cell Nanosized Components, Mechanics, and Diseases

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11.1



The Cell – Nanosized Components, Mechanics, and Diseases



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11.1.1 Cell Structure

Most of the biological cells are 1–100 μm in size and they comprise many nanosized

constituents (Fig. 11.2). Structural studies with nanoscale resolution can be performed by cryoelectron microscopy [11.7] (see Sect. 2.6), by electron microscopy

[11.13] of whole cells in liquid [11.8], or by far-field stimulated emission depletion (STED) optical microscopy [11.9, 11.14] (see Sect. 2.4). The interior of the

cell (Fig. 11.2) includes the cytoplasm and the nucleus which contains the chromosomes with the DNA strands carrying the genes and functions in the transmission

of hereditary information. The cytoplasm contains the cytosol which is the “fluid”

in the cell, the endoplasmic reticulum, microtubules, actin filaments, intermediate

filaments, mitochondria, and the Golgi apparatus. The actin filaments (ca. 8 nm in

diameter; see Fig. 11.3a) and the microtubules, in the shape of hollow tubes with

a diameter of ca. 25 nm, form the cytoskeleton. The ribosomes found in the cytoplasm with a diameter of ca. 25 nm (see Fig. 11.3a, b) are minute round particles

composed of RNA and protein which are active in the synthesis of proteins. The

ribonucleoprotein of the ribosome represents the last step of the gene expression



Fig. 11.2 (a) Schematic diagram of a typical eukaryotic cell which contains many nanoscale elements (see text). (b) The phospholipid bilayer membrane which covers the cell. (Reprinted with

permission from [11.6]. © 2003 Nature Publishing Group)



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Fig. 11.3 (a) Cryoelectron tomography of the slime mold cell Dictyostelium discoideum (image

815 nm by 870 nm by 97 nm) with subjectively added colors to mark actins filaments (reddish),

ribosomes (green), and membranes (blue) [11.7]. (b) The Haloarcula marismortui large ribosomal subunit [11.10]; RNA is shown in gray, the protein backbones are given in gold. The particle

is ca. 25 nm across. The macromolecular structures, such as the ribosome, that populate the cell

are functional nanostructures – “nanomachines” – with a much higher sophistication than that of

artificial nanostructures. (c) Density profile of a 2D projection of a 26 S proteasome in the cell

(a) derived from cryoelectrontomography. This molecular machine with a mass of 2.5 MDa has a

characteristic elongation of 45 nm [11.7]. (d–f) The shapes of different biological membranes can

be visualized by labeling them with fluorescently tagged proteins [11.12]: (d) endoplasmic reticulum (green; 30–100 nm), (e) Golgi apparatus (red/yellow; 30–100 nm), and (f) vesicular membrane

carriers (green; spheres with a diameter of 40–100 nm) for transport tasks from Golgi complex to

the plasma membrane. (g) Electron tomography of a neuronal filopodia (green) whose tip is in

close contact with an axonal bouton (blue) in the adult mouse hippocampus [11.12]. (Reprinted

with permission from [11.7] (a) (c), [11.10] (b), and [11.12] (d–g). © 2002 AAAS (a) (c), © 2000

AAAS (b), and © 2007 Nature Publishing Group (d–g))



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pathway where the genomic information encoded in messenger RNAs is translated

into protein. The large ribosomal subunit (Fig. 11.3b) includes the activity that catalyzes peptide bond formation – peptidyl transferase – and the binding site for the

G-protein factors that assist in the initiation, elongation, and termination phases of

protein synthesis [11.10].

A striking example of a large protein complex residing in the cytosol is the proteasome [11.11] (Fig. 11.3c) with proteolytic activities, responsible for degrading

proteins that have been marked for destruction by the attachment of a small ubiquitin protein molecule. A mitochondrion (see Fig. 11.2a) is a membrane – bound

organelle, about the size of a bacterium (500 nm in diameter and 1000 nm in length)

that carries out oxidative phosphorylation and produces most of the ATP in eukaryotic cells. The endoplasmic reticulum is a membrane network (Figs. 11.2a, 11.3d

and 11.4) within the cytoplasm where lipids, membrane-bound proteins, and secretory proteins are synthesized. Individual structural elements of the endoplasmic

reticulum of a cell can be imaged optically by the STED technique (see Sect. 2.4)

with a lateral resolution of <50 nm inside the living cell [11.9, 11.14] (Fig. 11.4).

From the endoplasmic reticulum proteins and lipids are transferred to the Golgi

apparatus (Figs. 11.2a and 11.3e), a network of stacked membranous vesicles, where

they are modified and sorted. Vesicular membrane carriers (Fig. 11.3f) can perform

transport tasks from the Golgi apparatus to the plasma membrane. Other cellular

nanostructures are the protrusions of nerve cells (Fig. 11.3g) to make junctions with

adjacent cells.

The cell is enclosed by a phospholipid bilayer membrane (Fig. 11.2b), with

hydrophilic heads and hydrophobic tails whose mechanical rigidity is altered by

the presence of integral protein molecules and cholesterol. Transmembrane protein

receptors, such as integrins, provide links between the extracelluar matrix (ECM)

and the cell interior.



Fig. 11.4 Optical image of the endoplasmic reticulum, of a living mammalian cell making use of

the stimulated emission depletion (STED) technique. Tubules as small as 60 nm can be seen (scale

bar, 500 nm). (Reprinted with permission from [11.9]. © 2008 National Academy of Sciences

USA)



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11.1.2 Mechanics, Motion, and Deformation of Cells

Cells undergo mechanical deformation when subjected to external forces [11.4]. For

the cell motion during cell migration contractile forces must be generated within the

cell [11.15]. In addition, cells can signal stress, such as endothelial cells living in

the interior walls of blood vessels, as they alter the expression of “stress-sensitive”

genes in response to shear flow in the blood (see [11.6]). The deformability of a cell

is determined largely by the cytoskeleton yielding an effective elastic modulus of

102 –105 Pa, orders of magnitude smaller than that of metals or ceramics (∼1011 Pa).

Experimental techniques for studying the mechanical behavior of cells are shown

in Fig. 11.5. In addition to atomic force microscopy (AFM; see Sect. 2.2), magnetic

twisting cytometry (MTC) is employed, where magnetic beads attached to a cell in

a magnetic field can deform a cell locally. In the micropipette aspiration experiment,

a cell is deformed by applying a suction through a micropipette on the cell surface.

In the case of optical tweezers of a laser trap (Fig. 11.5) a force is created between

a dielectric bead of high refractive index attached to a cell and a laser beam, pulling

the bead toward the focal point of the trap.

Most living cells generate or sense forces. Skeletal, heart, and smooth muscle

cells generate contractile forces on excitation, performing many essential functions

of the body. Endothelial cells can recognize the magnitude, type, and duration of

applied shear flow and respond accordingly (see [11.6]) in a healthy endothelium

or in vascular diseases. Fibroplast cells “crawl” like an inchworm by pulling the

cell body forward using contractile forces. These forces are generated by motor

proteins, such as kinesin, myosin, or dynein, powered by the hydrolysis of adenosine

triphosphate (ATP). It is not well understood how cells sense mechanical forces and

deformation and how they convert such signals to biological response (see [11.6]).



Fig. 11.5 Schematic representation of techniques for probing the mechanical properties of living cells. (a) Atomic force microscopy (AFM) and (b) magnetic twisting cytometry can probe

cell components at a force resolution of 10−10 and 10−12 N, respectively, and a displacement

resolution of less than 1 nm. (c) Micropipette aspiration (MA) and (d) optical trapping or optical

tweezer stretching are techniques which can deform an entire cell at a force resolution of 10−10 and

10−11 N, respectively. (Reprinted with permission from [11.6]. © 2003 Nature Publishing Group)



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Studies by modeling of the mechanical properties of a cell should exceed

continuum-based models and include the cytoskeleton, the viscous cytoplasmic

fluid, as well as the granularity and the heterogeneity of the cell.

The ability of cell membranes to adopt a great variety of shapes is one of the most

striking and intriguing properties of cells. Computer simulations indicate that the

forces necessary for this shaping arise from the concerted efforts of many proteins,

integrated within the membrane, mediated by the 4.7–5.2 nm thick lipid bilayer

that forms the membrane matrix and resists strong bending [11.12, 11.16]. In the

cell there is a great variety of membrane shapes: plasma membranes are practically flat; the cylindrical membrane structures of the tubules 30–100 nm in diameter

are strongly curved; the membrane structures that mediate endocytosis (the uptake

into the cell from outside) and exocytosis (the export of material from the cell),

and those that contribute to intracellular trafficking, form spheres of 40–100 nm

diameter. In addition, saddle-like shapes are found at membrane necks and junctions as well as in the stacking-up of the membrane disks of the Golgi apparatus

(see Fig. 11.3). Furthermore, these membranes undergo relentless shape transformations involving strong deformations and, in many cases, drastic rearrangements

of membrane structures, leading to their fusion and fission.

The large-scale curving of a membrane requires the concerted effort of many

membrane proteins forming domains. The physics of these long-range forces is a

crucial open question. The reason for these forces is the interaction of individual

membrane proteins which reduces the elastic energy and restricts the entropically favored thermal undulations of the membrane. Computer simulations showed

[11.16] that the net interaction of the protein is attractive and that the attraction

between proteins with sufficient intrinsic curvature is strong enough to cause them

to cluster in domains giving rise to a driving force for membrane shaping in real

biological conditions.



11.1.3 Cell Adhesion

When white blood cells fight viruses or bacteria or in the formation of metastases

of cancerous cells, cell adhesion and cell mechanics play an important role [11.17–

11.21]. In living cells, adhesion structures additionally have the ability to grow and

strengthen under force. The adhesion is mediated by a selective bonding of ligands

on one cell and receptors on an adjacent cell. The adhesion process is due to the

formation of domains (ca. 1 μm in size) of ligand-receptor pairs.

A nanoscale modeling of cellular adhesion has been studied experimentally by

the interaction of test vesicles containing arginine–glycine–asparagine (RGD) peptides as ligands with membranes containing integrin receptors as target cells [11.18,

11.19] (see Fig. 11.6a). Adhesion occurs by micrometer domains of agglomerated

ligand-receptor pairs (see [11.18]). These domains are separated by non-adhering

areas. This can be observed by reflection interference contrast (RIC) microscopy,

yielding a vertical resolution of 5 nm and a lateral resolution of 50 nm [11.19].

When both ligands and receptors are mobile within their membranes, strengthening



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Fig. 11.6 (a) In a model of cell adhesion, vesicles containing arginine–glycine–asparagine (RGD)

ligands are used as test cells whereas a membrane with integrin receptors on a substrate is employed

as target cell [11.18]. (b) Adhesion domains (enclosed in white) as seen by optical reflection interference contrast (RIC) microscopy in a vesicle interacting with a membrane [11.19]. (Reprinted

with permission from [11.18] (a) and [11.19] (b). © 2006 Wiley-VCH (a) and © 2008 National

Academy of Sciences USA (b))



is aided by lateral movement of intact bonds as a transient response to force-induced

membrane deformation [11.19].

A theoretical treatment has been given by calculating the total free energy of

the vesicle-membrane system, including the elastic deformation energy, the binding

energy of the ligand–receptor pair ensemble, and the positional entropy of mobile

RGD ligands and integrin receptors [11.19]. The calculations show a strengthening

of the adhesion domains under force as demonstrated in analogous experiments.

In the model study, the application of force leads simultaneously to a decrease in

the contact zone area and an increase in the adhesion energy density [11.19]. The

identification of this phenomenon in living cells would be desirable.



11.1.4 Disease-Induced Alterations of the Mechanical Properties

of Single Living Cells

This can be investigated by measuring the elastic properties at pN-level forces

[11.22, 11.23] which could provide insight into the progression of diseases. The

two examples of elastic measurements on human pancreatic cancer cells (Panc-1)

and on human red blood cells (RBCs) infected with the malaria parasite Plasmodium

falciparum will be briefly discussed.



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Epithelial pancreatic cancer cells subjected to repeated tensile loading showed a

three-fold decrease of the elastic modulus upon treatment with sphingosylphosphorylcholine (SPC), a bioactive lipid that promotes antiapoptotic (against controlled

cellular death) effects in human blood components such as blood plasma and highdensity lipoprotein (HDL) particles. The modulus decrease can be ascribed to a

SPC-induced reorganization of the keratin network to perinuclear regions of the cell

(see Fig. 11.7a). The modulus decrease and the increased deformability of cancer

cells may increase the probability of cell migration to another site in the body, a

critical step in cancer metastasis.

The malaria disease state occurs when the malaria-inducing parasite leaves

the liver and invades red blood cells where it produces proteins that modify the

RBC membrane and cytoskeleton. Healthy RBCs repeatedly deform from their

biconcave shape to pass through small blood vessels or capillaries. In contrast,

RBCs parasitized by P. falciparum show an up to 10-fold increase in elastic stiffness (Fig. 11.7b), they adhere to vascular endothelium, and therefore,

sequester in microvasculature contributing to organ failure [11.22]. The stiffening may arise from the perturbation of the attachments between the membrane

and the cytoskeleton. These attachments are facilitated by chemical interactions

involving ankyrin and the RBC anion transporter as well as protein 4.1 and

glycophorin A (see [11.22]). Alterations to this delicate molecular architecture

due to abnormalities that mediate cross-linking of cytoskeletal proteins can result



Fig. 11.7 (a) Treatment of human pancreatic cancer cells (Panc-1) with sphingosylphosphorylcholine (SPC) over a period of 60 min. causes significant reorganization of the keratin filaments

to perinuclear regions which may be the reason for a three-fold decrease of the cellular elastic

modulus. (b) Optical tweezers stretch a healthy red blood cell (top row) and a cell in a late stage

of infection with the malaria parasite (bottom row). The parasite is visible in the centers of the

bottom row images. Left column: tensile stretching of the cell by optical tweezers at a constant

force of 68 ± 12 pN; right column: stretching at a constant force of 151 ± 20 pN. (Reprinted with

permission from [11.22]. © 2005 Elsevier)



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in severe stiffening of the RBCs. By the P. falciparum parasite, several parasite proteins are introduced into the RBC membrane and cytoskeleton, altering

the mechanical response and the adhesive properties. Among these proteins,

P. falciparum ring-infected erythrocyte surface antigen (RESA) or Pf 155 gets

deposited into the cytoplasmic surface of the erythrocyte membrane from the

dense granules in the apical region of the merozoite (parasitic protozoans which

contribute to the proliferation of P. falciparum) during parasite invasion. It has

been speculated that RESA interaction with the spectrin network underneath the

RBC membrane contributes to the increased elastic modulus of parasite harboring

RBCs (see [11.22]).

The structural and mechanical modifications of red blood cells (RBCs) due to

parasitization by malaria-inducing P. falciparum can be extracted from optically

measured maps of the refractive index and of nanoscale cell membrane fluctuations [11.24]. These findings may provide information on pathological states that

cause or accompany human diseases. Furthermore, disease mutations in human

intermediate filament (IF) proteins in the cell nucleus and in the cytoplasma indicate [11.25] that the nanomechanical properties of cell-type-specific IFs are central

to the pathogenesis of diseases as diverse as muscular dystrophy and premature

aging.

Theoretical and computational approaches, taking into account the data of

advanced experimental techniques, may provide new insight into the connection

between cell mechanics and function [11.26]. For these approaches the consideration of a wide range of length scales is of importance. For treating the overall

mechanical response of individual cells, continuum-based computational models

are favorable, whereas for nanostructural modeling molecular dynamics (MD)

techniques are appropriate.



11.1.5 Control of Cell Functions by the Size

of Nanoparticles Alone

To date, about 10 years after the regulatory approval of liposomaly encapsulated doxorubicin to treat various forms of cancer, no such “higher functionality” nanoparticle has reached the clinic yet. The reason is because it

remains difficult to identify and validate biological targets that are specific

enough for cancer cells [11.27]. New results [11.28] point to the importance

of size as both a bioactive agent and an agent of toxicity. It was found that

the uptake of Au or Ag nanoparticles (2–100 nm) decorated with Herceptin –

a drug that binds Erb B2 receptors on breast cancer cells – was size dependent [11.28]. Most importantly, specific cell-growth pathways were inhibited and

programmed cell death (apoptosis) was enhanced, particularly for particles in

the 40–50 nm range.



11.2



Nanoparticles for Bioanalysis



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11.2 Nanoparticles for Bioanalysis

Nanoparticles are used for efficient and selective tagging of a wide range of biologically and biomedically important targets, such as individual biomolecules, bacteria,

and cancer cells. Metallic, semiconductor, oxide, or magnetic nanoparticles are used

[11.29–11.35]. The further development of these nanoparticles with much higher

photosensitivity and much higher stability against photobleaching than conventional organic dyes will provide a variety of advanced tools for molecular biology,

genomics, proteomics, drug delivery, as well as diagnosis and therapy of infectious

diseases and cancer.



11.2.1 Various Materials of Nanoparticles

One of the most widely used nanoparticle (NP) materials is Au-NPs as colorimetric

probe due to the size and shape-dependent surface plasmon excitation (see Sect.

7.6) which changes upon aggregation with other molecules. Ultrasensitive analysis

of oligonucleotides, proteins, and other biomolecules has been achieved using AuNP biomarkers which are already commercial products [11.35]. One example is

the lateral flow strip for fast pathogen detection and point-of-care diagnosis [11.36,

11.37].

Semiconductor nanoparticles of CdSe, CdTe, CdS, ZnSe, etc. [11.31, 11.35]

with diameters of 2–10 nm and wide absorption bands provide bright (10–20-fold

higher than an organic fluorophore) and narrow-fluorescence emission bands

(see Fig. 11.8). The resistance of the semiconductor nanoparticles against photobleaching compared to the fast bleaching of organic dyes is demonstrated

in Fig. 11.9a.

Silica nanoparticles with dye doping and high analytical sensitivity can be fabricated by a precursor technique that, e.g., integrates fluorescein isothiocyanate

(FiTC) into the 3-amino propyltriethoxysilane precursor for a final incorporation

of the dye into the silica matrix [11.38]. Besides single-dye doping, multiple-dye

incorporation into silica NPs can be performed (see [11.35]).

Magnetic nanoparticles of, e.g., iron oxide (Fe3 O4 , γ-Fe2 O3 ) are of biological

and clinical interest (see [11.39]) since they are fully biocompatible because the

human body already contains around 3–4 g Fe in the form of ferritin, hemoglobin,

etc. These nanoparticles are superparamagnetic, i.e., they magnetize strongly in

an applied magnetic field but retain no permanent magnetization once the field is

removed. This magnetic behavior may improve drug delivery by literally dragging

therapeutic agents attached to these nanoparticles to specific areas in the body under

the influence of an external magnetic field (see Fig. 11.10). In addition, iron oxide

nanoparticles have already been approved for clinical use as magnetic resonance

imaging (MRI) contrast agents. These agents work by altering the relaxation rates



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a



b



c



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Biology on the Nanoscale



11.2



Nanoparticles for Bioanalysis



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Fig. 11.9 Resistance of CdSe/ZnS nanoparticles to photobleaching and their ability of multicolor

labeling. (a) Top row: antigens in a 3T3 cell nucleus were labeled with CdSe/ZnS streptavidin nanoparticles (630 nm emission, red) and microtubules were labeled with the organic dye

Alexa Fluor 488 (488 nm emission, green); bottom row: microtubules labeled with the CdSe/ZnS

nanoparticles (red) and nuclear antigens stained with Alexa 488 (green). The nanoparticles resist

to photobleaching under continuous illumination [11.41]. (b) Pseudocolored image with nanoparticle staining in fixed human epithelial cells: Cyan (655 nm) corresponds to the nucleus, magenta

(605 nm) labels Ki-67 protein, orange (525 nm) labels mitochondria, green (565 nm) labels

microtubules, and red (705 nm) labeling actin filaments [11.31]. (Reprinted with permission from

[11.41] (a) and [11.31] (b). © 2003 Nature Publishing Group (a) and © 2005 Nature Publishing

Group (b))



of water protons (see Sect. 12.2) that are trying to realign with a static magnetic field

following the application of radiofrequency (RF) pulses. The iron oxide nanoparticles affect the transverse relaxation times (T2) leading to negative contrast or dark

spots on the T2-weighted MR images.



Fig. 11.8 Comparison of the absorption (ABS) and emission (EM) of the common organic

dye rhodamine red and the genetically dye-encoded DsRed2 protein with that of six different

sized ZnS-coated CdSe nanoparticles (a,b) [11.40]. (c) Photo of the size-tunable fluorescence

and spectral range of the six CdSe/ZnS nanoparticles dispersions of (b). (d) Comparison of the

size of the CdSe/ZnS nanoparticle (r ∼3.0 nm; emitting at 555 nm) surface-functionalized with

dihydrolipoic acid (red shell, ∼1.0 nm) with that of a midsize maltose binding protein (MBP,

mass ∼44 kDa, 3×4×6.5 nm3 ) [11.31]. (Reprinted with permission from [11.40] (a) (b) and

[11.31] (c) (d). © 2000 National Academy of Sciences USA (a) (b) and © 2005 Nature Publishing

Group (c) (d))



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