Live Cell Microscopy: A Physical Chemistry Approach - The Journal of

Subscriber access provided by READING UNIV

Feature Article

Live Cell Microscopy: A Physical Chemistry Approach Somen Nandi, Surajit Ghosh, and Kankan Bhattacharyya J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b11689 • Publication Date (Web): 01 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Physical Chemistry

Live Cell Microscopy: A Physical Chemistry Approach Somen Nandi1, Surajit Ghosh*2,3 and Kankan Bhattacharyya*4

1

Department of Physical Chemistry, Indian Association for the Cultivation of Science,

Jadavpur, Kolkata 700 032, India 2

Organic & Medicinal Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4,

Raja S. C. Mullick Road, Jadavpur, Kolkata-700032, West Bengal, India.Fax: +91-33-24735197/0284; Tel: +91-33-2499-5872. 3

Academy of Scientific and Innovative Research (AcSIR), CSIR-Indian Institute of Chemical

Biology Campus, 4 Raja S. C. Mullick Road, Kolkata 700 032, India. 4

Department of Chemistry, Indian Institute of Science Education and Research Bhopal,

Bhopal- 462 066, Madhya Pradesh, India

CORRESPONDING AUTHORS INFORMATION: *

E-mail: [email protected], [email protected]

1 ACS Paragon Plus Environment

Fax: (91)-33-2473-2805

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Probing dynamics of intracellular components using physical chemistry techniques is a remarkable bottom-up approach for understanding the structures and functions of a biological cell. In this feature, we give an overview on local polarity, solvation, viscosity, acid-base property, red-ox processes (thiol-disulfide exchange) and gene silencing at selected intracellular components inside a live cell. Significant differences have been observed between cancer cells and its non-cancer counterparts. We demonstrate that thiol-disulfide exchange, calcium oscillation and gene silencing are manifested in time dependence of fluorescence intensity. We show that fluorescent gold nano-clusters may be used in drug delivery (e.g. doxorubicin) and selectively killing of cancer cells. Further, we discuss dynamics and structural changes of DNA quadruplexes and i-motifs, induced by different external conditions (e.g. pH) and additives (e.g. K+ and other target specific small molecules). We demonstrate that peptidomimetic analogues have high specificity over double-stranded DNA for binding with i-motifs and G-quadruplexes. These results may have significant biological implications.


Page 2 of 45

Page 3 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Understanding intracellular components and their dynamics using physical chemistry approach is challenging and at the same time highly rewarding. Capturing the dynamic complex environment inside a live functional cell is one of the longstanding goals of biophysical chemistry.1 This has inspired a large number of recent studies on live cells in vivo using electron microscopy and super-resolution fluorescence microscopy.1-11 In this article, we discuss about the recent applications of a confocal microscope12-14 to unravel various interesting phenomena occurring in a live cell. A biological cell is ~50-100 times larger than the spatial resolution (/2 ~200 nm, Abbey Limit) of a confocal microscope. Many fluorescent probes selectively localize in a specific region or organelle of a cell. The size of a fluorescent dye is in the order of ~1 nm and hence, they report on a region of radius ~1 nm around the centroid of the probe. We have earlier demonstrated that it is possible to probe different regions within a micelle of diameter ~20 nm, immobilized in a tri-block co-polymer gel.15 The immobilized micelles and gels (formed by pluoronic P123 and F127) contain a hydrophobic core of polypropylene oxide (PPO) and a hydrophilic corona of polyethylene oxide (PEO) (Scheme 1A).15 We determined translational diffusion coefficient (Dt) of fluorescent dyes in these gels by fluorescence correlation spectroscopy (FCS). According to the Stokes-Einstein relation, Dt is related to size (radius, rH) of the diffusing particle and local viscosity (η) as

Dt 

k BT 6rH

(1)

In such a gel, the highly hydrophobic probe DCM (Scheme 1B) resides in the PPO core of the immobilized micelles, while the anionic and hydrophilic dye, coumarin 343 (C343, Scheme 1B) remains preferentially in the peripheral corona region, in contact with the bulk water. The localization of the two probes over a small region is confirmed by red-edge



Page 4 of 45

A Hydrophilic corona (PEO block) Pore (“Void”) Hydrophobic core(PPO block)

-

B

O3S

OH

NC

COON

O

O

O

N

C

CN

CH3 N CH3

O -

CF3

SO3-

O3S

HPTS

C343

H3C

O

C C H H

C153

DCM O N

F N

F

O

N

S O

O

N H

F

H N

O

F O

N

F

O

ER-Tracker Blue-White DPX

C

H

N O

O

S Protein

O N

N

Cysteine

O

CPM

O

O

O

HS Protein Cysteine

N

Free CPM, Non-fluorescent

O

O

Bound CPM, Fluorescent

Scheme 1. (A) Representative picture of micelle and gel formed by pluoronic P123 and F127; Adapted with permission from ref. 15, Copyright 2009 John Wiley and Sons; (B) Structure of different dyes and (C) labeling reaction scheme of thiol group of proteins by CPM. 4 ACS Paragon Plus Environment

HS Protein

Page 5 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


excitation shift16,17 of emission maxima.15 In the immobilized micelles, Dt of both the dyes is much smaller than those in bulk water indicating higher friction (i.e. viscosity). However, the magnitude of increase in local viscosity in the gel, sensed by the two probes, is different. In F127 gel, Dt of DCM is about ~300 times smaller than that in bulk water which implies an about 300 times higher viscosity in the PPO core compared to the bulk water.15 Dt of C343 at the corona region in F127 gel is ~20 times smaller than that in bulk water, indicating about 20-fold higher viscosity. Thus, compared to the corona the local friction (viscosity) of the core is ~15 times higher.15 Encouraged by this result, we have explored different regions of a live cell using nano-metric fluorescent probes localized in specific locations of a cell. We have attempted to map local polarity, solvation, viscosity, acid-base property, red-ox processes (thiol-disulfide exchange) and gene silencing inside a live cell. In many cases, we have compared a cancer cell with their corresponding non-cancer counterpart. We observed remarkable differences between cancer and non-cancer cells. These results eventually prompted us to explore drug delivery and selective killing of cancer cells. In this article, we will discuss and highlight some of our recent findings.

2. Probing Selected Regions of a Cell 2.1. Local Solvation Environment at Selected Regions of a Live Cell The local solvation environment (polarity or dielectric constant, ) controls many complex biological processes or events.18 For instance, transport of an ion or a proton (i.e. acid-base properties) or an electron requires stabilization of the charged and polar species, through solvation. There are many fluorescent probes whose fluorescence maximum depends on local polarity and solvation energy.16-18 The local polarity may be estimated by labeling different organelles of a live cell with a solvent sensitive fluorescent probe, recording



fluorescence spectra under a confocal microscope and then comparing those with the emission spectra of the same dye in simple solvent.19,20 For a Chinese hamster ovary cell, we used the highly hydrophobic dye, DCM for the cytoplasm region and DAPI (4′,6-diamidino-2-phenylindole), a DNA binding probe molecule for the nucleus (where the DNAs reside).21 We found that the emission maximum of DCM at cytoplasm resembles that in 2-propanol with dielectric constant of  = 15. On the contrary, for DAPI which resides in the highly polar nucleus having a lot of ions (phosphate groups of DNA and counter ion), the local polarity (emission maximum) is similar to that in 1:1 methanol-water mixture with  = 65.21

Figure 1. Chinese hamster ovary cell stained by C153 dye: (A) Confocal image; (B) Emission spectra; (C) Solvation dynamics (decays of solvent correlation function, C(t)).22 Reprinted with permission from ref. 22. Copyright 2013 American Chemical Society. Cytoplasm contains large number of different organelles with different polarity. Using coumarin 153 (C153, Scheme 1B) as a fluorescent probe we could visualize cytoplasm, lipid droplets and nucleus of a Chinese hamster ovary cell (Figure 1).22 The lipid droplets appear as bright dots (size ~800 nm,22 Figure 1A). The emission maximum of C153 in the nucleus, cytoplasm and lipid droplets are observed at ~525 nm, 521 nm and 502 nm, respectively (Figure 1B). From the emission maximum of C153 in different solvent, the polarity () of nucleus, cytoplasm and lipid droplets are estimated to be ~26, 22 and 7, respectively.22 In the 6 ACS Paragon Plus Environment

Page 6 of 45

Page 7 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


case of nucleus of the same cell, DAPI reports a much higher polarity ( = 65)21 because of the following reasons. DAPI localizes at the minor groove of the double-strand DNA i.e. near the ions (phosphate groups of DNA and counter ions).23 C153 is a hydrophobic dye which resides in hydrophobic pockets inside the nucleus. The different polarity reported by two dyes inside the nucleus of the same cell reveals the variation of polarity inside a small part of the cell. There is tremendous recent interest to understand solvation dynamics of “biological water” near a biological systems like protein, DNA etc.24-28 It is observed that solvation dynamics of biological water contain a component of ~100-1000 times slower than that in bulk water (~1 ps).24-29 Until recently, biological water are referred to those in an aqueous solution of protein, DNA or other bio-molecules. The real biological water, however, resides inside a live cell.30 Using time resolved confocal microscopy, we have been able to capture solvation dynamics of real biological water at different locations within a live cell (Table 1).21,22,30-35 Though the ultrafast femtosecond component of solvation dynamics inside a live cell is still elusive, the experiment with picoseconds time resolution reveals a component in ~100-1000 ps time scale in the live cell. In the case of Chinese hamster ovary cell stained by C153, the average solvation time ⟨τs⟩ in various region follows the order as lipid droplets (~3600ps) > cytoplasm (~1100 ps) > nucleus (~750 ps) (Figure 1C and Table 1).22 We found that the Endoplasmic Reticulum (ER) tracker dye, which localizes at the ER region, is itself a solvation probe. We have used this probe to monitor the solvation dynamics at the ER-region of lung cells both in cancer (A549) as well as in non-cancer (WI38) cells (Table 1).35



Page 8 of 45

Table 1. Emission maxima and average solvation times (⟨τs⟩) in different regions of a cell stained by different fluorescent probes.21,22,30-35 System

Emission

⟨τs⟩a in ps

Dye

Region

maxima (nm)

CPM

Membrane

476

500

C153

Cytoplasm

521

1100

C153

Nucleus

525

750

C153

Lipid Droplets

502

3600

CPM

Lipid Droplets

476

1600

C153

Lipid Droplets

495

3750

Lung

CPM

Cytoplasm

468

250

cancer

DAPI

Nucleus

460

300

C153

Cytoplasm

533

850

ER

Endoplasmic

Tracker

Reticulum

510

250

CPM

Lipid Droplets

472

800

C153

Lipid Droplets

505

1700

Non-cancer

CPM

Cytoplasm

474

400

lung

DAPI

Nucleus

460

450

(WI38)

C153

Cytoplasm

525

1050

ER

Endoplasmic

Tracker

Reticulum

500

1000

470

1400

475

850

Cell Line Chinese hamster ovary

(A549)

Breast Cancer

CPM

(MCF7) Non-cancer Breast (MCF10A) a

CPM

Mitochondria (Perinuclear clustering)

Mitochondria (discrete)

± 50 ps


Page 9 of 45 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. Lipid droplets inside lung cells. (A) WI38 labeled by CPM; (B) A549 labeled by CPM; (C) A549 labeled by nile red; (D) merged image of (B) and (C).31 Reprinted with permission from ref. 31. Copyright 2015 American Chemical Society.

2.2. Lipid Droplets and Warburg Effect: Cancer vs Non-cancer Cells The accelerated rate of glycolysis in cancer cell and suppression of degradation of pyruvate leads to the formation of fat.36-39 This results in proliferation of lipid droplets (“fat depot”) in cancer cell compared to normal cell. This is referred to as Warburg effect.36-39 In fact, inhibition of the synthesis of lipid droplets have been suggested to be a therapeutic cure of cancer.39 We have compared the number of lipid droplets in a cancer cell with that in a normal (non-malignant) cell.31,32 The lipid droplets can be non-covalently labeled by C153 or nile red.22,32 The lipid droplets appear as bright dots inside the cell (Figure 2). Comparing many cancer and non-cancer cells, we found that the number of lipid droplets in a cell is a 9 ACS Paragon Plus Environment


good diagnostic measure in identification of cancer cell. In a normal cell, the number of such lipid droplets is below 15 (

Live Cell Microscopy: A Physical Chemistry Approach - The Journal of

Recommend Documents