Cellular Nanostructures and Their Investigation.

History and Perspectives

C. M. Niculiìe1,2, A. O. Urs1, E. Fertig1, C. Florescu1, M. Gherghiceanu1, M. Leabu1,2

1 “Victor Babeè” National Institute of Pathology, Department of Cellular and Molecular Biology, Bucharest, Romania
2 “Carol Davila” University of Medicine and Pharmacy, Division of Histology, Cellular and Molecular Biology, Bucharest, Romania

Abstract— Qticpk|cvkqp" qh" dkqoqngewngu" kpukfg" qt" qwvukfg" Chemistry, but again the distinction was jointly shared with
vjg"egnn."cv"vkuuwg"ngxgn."rtqxgf"vq"tgurgev"c"oketq/"cpf1qt"pcpq/ other two personalities (Eric Betzig and William E.
oqtrjqnqi{" cpf" vjgkt" hwpevkqpu" ctg" fgrgpfgpv" qp" vjg" tkijv" Moerner) “for the development of super-resolved fluores-
uvtwevwtkpi" qh" vjgug" oketq/" qt" pcpq/uvtwevwtgu0" Vjku" ujqtv" cence microscopy” [5]. It is as an arch over time: two Ro-
paper summarizes the history of cellular nanostructures’ study
manian scientists with two significant contributions, by
htqo" vjgkt" fkueqxgt{." vq" ewttgpv" ghhqtvu" kp" fgekrjgtkpi" vjgkt"
hwpevkqpu."ceeqtfkpi"vq"vjg" jkij"eqorngzkv{"qh"nkxg"egnn"qticpk/ developing approaches for cell study (on fixed and live
|cvkqp0" Yg" yknn" cfxqecvg" vjcv" vjg" fggrgpkpi" qh" qwt" mpqyngfig" cells) with a 40 years interval in-between.
kp"egnn"dkqnqi{"jcu"tgswktgf"cpf"yknn"hwtvjgt"pggf"c"ykug"eqodk/ In this paper, we will point out some of the cornerstones
pcvkqp"qh"ghhqtvu"ocfg"d{"dkqnqikuvu."ejgokuvu."rj{ukekuvu."ogfk/ of progress in cellular nanostructure study obtained through
ecn"fqevqtu"cpf"gpikpggtu0" methodological and technological advances due to com-
Keywords—" gngevtqp" oketqueqr{." UVGF" oketqueqr{." egp/ bined efforts of biologists, chemists, physicists, medical
vtkhwicvkqp."gngevtqrjqtguku."cvqoke"hqteg"oketqueqr{0" doctors and engineers.

II. HISTORY OF CELLULAR NANOSTRUCTURE STUDY

I. INTRODUCTION
We may consider the start for cellular nanostructures in-
Cellular nanostructure identification began long before vestigation to be due to EM use in biology. But EM allows
the term was introduced in literature. This happened at the scientists to describe the internal morphology of a cell, with
middle of the 20th century through the contribution of no information about functions. Other methods were neces-
George Emil Palade, a Romanian born scientist, who, to- sary to assess the functions of elements in the organization
gether with Albert Claude, succeeded in setting up the use of of a cell (e.g. organelles). These methods involved centrifu-
electron microscopy (EM) in cell/organelle study [1-3]. In gation to isolate and purify morphological elements of cells
1974 the Nobel Prize in Physiology or Medicine was award- and a plethora of biochemical investigations to describe the
ed jointly to three scientists (the two mentioned above, to- composition of purified components and to decipher their
gether with Christian de Duve), ”for their discoveries con- functions.
cerning the structural and functional organization of the
cell” [4]. Soon thereafter, the detailed morphological organ- A. Electron microscopy
ization of the cell interior was deciphered, membrane
The progress contributed by EM to cell investigation was
bounded organelles were described (micro-structures) and
a morphological one, allowing visualization of the ultra-
even ribosomes were identified and discovered to be the
structure at the nano level. This improvement was due to a
machine that produce proteins. As a matter of fact, the ribo-
specific double fixation procedure, meaning a fixation of the
some was the first identified nano-structure. Nowadays, we
proteins by glutaraldehyde (sometimes mixed with formal-
may define cellular nanostructures as morphological, com-
dehyde [6]) and a postfixation step using a buffered osmium
plex elements in the living organisms with dimensions in the
tetroxide solution [3], targeting unsaturated fatty acids in
range of nanometres, in all three spatial directions (e.g.
membrane lipids. Using this double fixation both proteins
ribosome, proteasome, apoptosome). For more than half a
and membrane lipids are retained in native positions in a
century cell biologists have traditionally described struc-
spatial relationship very close or identical to that in the liv-
tures, as elements in the organization of a cell seen under a
ing cell. Further on, embedding the tissue sample in a resin,
light microscope and ultrastructures as elements that can
ultrathin sectioning (about 60nm thick sections), and con-
only be observed by EM. However, the scientific and tech-
trasting by lead citrate (for phosphate groups, mainly in
nological progress at the beginning of the third millennium
membrane phospholipids, sulfhydryl and carboxyl groups in
will change the above mentioned convention. Stefan W.
various substrates [7]) and uranyl acetate for phosphate
Hell pushed the resolution of light microscopy beyond the
groups in nucleic acids create detailed images of cell mor-
limits imposed by Abbe’s equation. Again, a Romanian born
phology under EM. Therefore the conservation of mem-
scientist received, in 2014, the Nobel Prize, this time in
branes proved to be critical in the investigation of cell mor-

© Springer International Publishing AG 2017 337

S. Vlad and N.M. Roman (eds.), International Conference on Advancements of Medicine and Health Care through Technology;
12th - 15th October 2016, Cluj-Napoca, Romania,
IFMBE Proceedings 59,
DOI: 10.1007/978-3-319-52875-5_70
338 C. M. Niculiìe et al.

phology, revealing the trilaminar ultrastructure of the plas- a better understanding of the liquid chromatography utility
ma membrane, and membrane bounded organelles. The in protein investigation, the readers can see [17].
three laminae of any biomembrane are evidenced as two It is noteworthy that all biochemical approaches require
electron-dense (mainly stained by lead at the polar heads of equipments that have continuously improved in their per-
the membrane phospholipids) defining in-between the third formances, by the common efforts of experts in cell biology
one that is electron-lucent and represents the part of the and engineers.
membrane bilayer containing lipid hydrophobic tails. Simul-
taneously with the description of the morphological ele- III. CURRENT APPROACHES
ments, the interest was focused on deciphering their func-
tions. To accomplish this task, it was necessary to isolate In the past, the progress in our knowledge about the cell
and purify the identified ultrastructures. was achieved by methods addressing fixed or disintegrated
cells. However, the real challenge for scientists has begun to
B. Centrifugation be studying biological mechanisms in live cells. To do this,
experts in biology, medicine, chemistry, physics and engi-
To isolate and purify the organelles, cells have had to be neering have to join their efforts in order to find solutions to
homogenized and various cellular fractions had to be sepa- accomplish this goal. In the next sections, we will consider
rated by (ultra)centrifugation. A coarse isolation was done some of these approaches, which push our knowledge closer
by differential centrifugation obtaining various fractions to the biological reality.
step-by-step, according to organelle density, but to obtain
purified fractions, (ultra)centrifugation in density gradients A. Atomic force microscopy
(discontinuous or continuous) was used [8-12]. The isolated
and purified morphological elements (organelles, plasma Atomic Force Microscopy (AFM) is a type of scanning
membrane vesicles, components of the cytoskeleton) were probe microscopy that was developed in the 1980s and has
biochemically analysed for (macro)molecular content to been used to visualize nanoscale cellular structures from
deduce and further on to prove specific functions. living cells. AFM operates by measuring the force between
a scanning probe (a sharp tip) and the sample. The tip is
C. Biochemistry located at the end of a cantilever and it scans the surface of
the sample, generating a three-dimensional image of the
Therefore, biochemical analysis of the cellular fractions surface. The force imposed on the sample is constantly regu-
helped cell biologists in deciphering organelles’ functions. lated by a feedback mechanism [18]. Because AFM doesn’t
The most useful techniques were electrophoresis and chro- require extensive sample preparation – like EM – and it also
matography. permits investigation of samples in aqueous solutions, bio-
Electrophoresis allowed the identification of different logical structures can be observed in their native environ-
charged macromolecules (mainly proteins and nucleic acids) ment.
by their various migration speed, led by an electric field, in a There are three main modes of operation in AFM: (i) con-
viscous medium (usually polyacrylamide or agarose gels). tact mode (the tip of the cantilever is in physical contact
The most used procedure was SDS-PAGE in a discontinu- with the surface of the sample [19]; it is mainly used for
ous buffer system [13]. An extension of this technique was rigid samples such as crystals, semiconductors and metals
represented by the extraction of protein bands from gels and [20], but also for living adherent cells [21]), (ii) tapping
their absorption on various membranes (nitrocellulose or mode (the cantilever oscillates at its resonance frequency; as
PVDF sheets) followed by specific identification of protein it approaches the surface of the sample, the interaction of
species by immunoblotting [14], a technique later referred to forces causes a decrease in the amplitude of the oscillation;
as Western blotting (WB), a term coined by W. Neal Bur- an image is produced by measuring the force of the intermit-
nette, in 1981 [15]. The method rapidly proved to be a very tent contacts between the tip of the cantilever and the sample
useful procedure to assess proteins in biological samples, surface; it is used for soft samples, like suspended mamma-
including organelles/nanostructures, and eliciting a great lian cells [22]) and (iii) non-contact mode (the cantilever tip
interest for the scientists in cell biology [16]. doesn’t come in contact with the sample, the image is gener-
A more accurate method, in terms of quantitative analy- ated by measuring the distance between the tip and the sam-
sis, was chromatography in a diversity of procedures, ex- ple at each data point [23]; it is also used for soft samples).
ploiting different types of molecular interactions in biologi- With the help of AFM, a cellular nanostructure involved
cal molecule identification and even isolation. Among these, in cell secretion was discovered in the late 1990s. Studies on
liquid chromatography (with standard or high performance live pancreatic acinar cells showed the presence of circular
approaches) was highly used, because it allowed separation pits in the apical membrane, which contained smaller de-
of (macro)molecules without any chemical degradation. For

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Cellular Nanostructures and Their Investigation. History and Perspectives 339

pressions involved in cell secretion [24]. The depressions preparation: (i) plunge freezing in a secondary cryogen for
were called porosomes and they have been identified in vitrifying small cells in suspension and (ii) high-pressure
other secretory cells as well [25-30]. freezing followed by cryo-sectioning (CEMOVIS), suitable
for cell suspensions and for tissue [38]. Before examination
B. STED microscopy
under cryo-electron microscope, the sample is transferred in
The resolution of conventional light microscopes is lim- a special holder (cryo-holder) where it is maintained at liq-
ited to approximately 250nm in the focal plane (xy) and uid nitrogen temperature.
450-700nm along the optical axis (z). In the past two dec- Cryo-electron tomography allows the analysis of cellular
ades however, a handful of so-called super-resolution tech- ultrastructure in three dimensions by acquiring a series of
niques have emerged to overcome the diffraction barrier, images taken around a tilt axis [39]. The final tomograms
notably: structured illumination microscopy (SIM), stimu- used for image processing and reconstruction of the intracel-
lated emission depletion (STED), photoactivated localiza- lular structures and protein complexes [40]. The direct elec-
tion microscopy (PALM) and stochastic optical reconstruc- tron detector device (DDD) cameras with underlying com-
tion microscopy (STORM) [31]. Whereas PALM or plementary metal-oxide semiconductor (CMOS) technology
STORM use sequential activation of fluorophores and sub- produce images of extraordinary high-quality that improve
sequent image reconstruction, STED microscopy relies on the results of digital image processing [41]. High-pressure
two synchronized lasers, one for the excitation of fluoro- freezing, combined with CEMOVIS and advanced software
phores and a doughnut shaped laser for de-excitation. When processing are the only way to visualize the complex molec-
superimposing the two lasers, the central region of the ular machine inside cells, at atomic scale, with Ångström
doughnut maintains fluorescence, thereby reducing the point resolution [42]. Future decades will make possible a mo-
spread function (PSF) and increasing resolution beyond the lecular atlas of cells at atomic resolution.
diffraction limit [32].
Since its inception, STED was successfully employed in IV. PERSPECTIVES
distinct areas of life-sciences to help resolve the nanostruc- In the future, improved communication between scientists
ture and dynamics of molecules, in some cases reaching in the fields of cell biology and engineers will be needed.
near-electron microscopy resolution. For example, STED The premises are good. Beyond the approaches mentioned
was used to visualize components of the nuclear pore com- under the previous section, other very promising techniques
plex at 20nm resolution, revealing the eightfold symmetry of seem to be successfully used in cell physiology and/or pa-
the protein gp210 [33]. STED microscopy was used to thology studies. For example, investigation of molecular
study the ultrastructure of endogenous F-actin in living interactions and morphology of cellular nanostructures at a
neurons, identifying actin lattices in both dendrites and ax- nanometre resolution can be done by small-angle X-Ray
ons [34]. STED helped understand the fate of synaptic vesi- solution scattering (SAXS), a technique carrying the ad-
cle proteins following their fusion with the plasma mem- vantage that the proteins do not need to be crystallized.
brane [35]. In another study, individual HIV virions could
be observed clustering at synapses formed between dendritic
ACKNOWLEDGEMENT
and T cells, in culture [36]. With super-resolution systems
becoming more accessible and easy to operate, one can Supported by ANCSI under Program NUCLEU, project
expect a surge of studies in the coming years shedding new numbers PN 16.22.02.02 and PN 16.22.03.02.
light on the inner workings of cells, at nanometre resolution.
C. Cryo-electron microscopy CONFLICT OF INTEREST
Cryo-electron microscopy in life sciences represents an The authors declare that they have no conflict of interest.
efficient technique which began to develop in the 1970s and
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with non-contact atomic force microscopy. Int J Mol Sci Corresponding author:
16(8):19936-19959 Author: Dr. Mircea Leabu
24. Schneider SW, Sritharan KC, Geibel JP, et al. (1997) Surface dy- Institute: “Victor Babes” National Institute of Pathology
namics in living acinar cells imaged by atomic force microscopy: Street: 99-101, Splaiul Independentei, sector 5
identification of plasma membrane structures involved in exocyto- City: Bucharest
sis. Proc Natl Acad Sci U S A 94(1):316-321 Country: Romania
Email: [email protected]

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