Cell biology is the study of cell structure, physiological properties and cell function. It involves the study of cell organelles, interactions between the cells and their environment, life cycle, division, and death. Cell biology is closely related to genetics, molecular biology, developmental biology, and biochemistry.

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Figure 1. The eukaryotic cytoskeleton. Actin filaments are shown in red, microtubules in green, and the nuclei are in blue. Staining for actin was done using rhodamine–phalloidin, for microtubulin using Alexa488-conjugated anti-alpha-tubulin stain, and for DNA, using Hoechst dye.

Inhibitors and activators are critical tools for researchers in the field of cell biology to obtain a comprehensive understanding of cell function, cell signaling and the intracellular mechanisms that control cell fate, function, and phenotype. Here we review commonly used inhibitors and activators for studying various aspects of cell biology for eukaryotic cells like secretion, adhesion, cytoskeletal dynamics, endoplasmic reticulum and Golgi. Another Labome article addresses endocytosis and its activators and inhibitors. Researchers have raised and discussed quality and specificity concern about small molecule inhibitors and activators [1], similar to the issues about antibody quality.

The cytoskeleton provides any cell with structure and shape. Eukaryotic cells contain three main kinds of cytoskeletal filaments: microfilaments, intermediate filaments, and microtubules. Commonly used inhibitors of cell cytoskeleton are listed in Table 1 and commonly used activators of cell cytoskeleton are listed in Table 2.

These are the thinnest filaments of the cytoskeleton. They are composed of linear polymers of actin subunits, and generate force by elongation at one end of the filament coupled with shrinkage at the other, causing a net movement of the intervening strand.

The average size of these filaments is 10 nanometers in diameter and are more stable (strongly bound) than actin filaments, and heterogeneous constituents of the cytoskeleton. Intermediate filaments organize the internal 3-dimensional structure of the cell, organelles and serving as structural components of the nuclear lamina. They also participate in some cell-cell and cell-matrix junctions.

They are hollow cylinders about 23 nm in diameter, most commonly comprising 13 protofilaments which, in turn, are polymers of alpha and beta tubulin. They have a very dynamic behavior, binding GTP for polymerization, and are commonly organized by the centrosome.

Other less commonly used microtubule inhibitors include ciliobrevin D [2], an inhibitor of the minus-end–directed microtubule motor cytoplasmic dynein, and combretastatin A4, an inhibitor of tubulin polymerization [3].

Endoplasmic reticulum (ER) is an organelle in eukaryotic organisms that forms an interconnected network of membrane vesicles. It is involved in the synthesis, modification, and transport of cellular materials. It extends from the cell membrane through the cytoplasm and connects continuously with the nuclear envelope. The functions of endoplasmic reticulum vary greatly depending on its cell type, cell function, and cell needs. It consists of two regions which differ in both structure and function.

It is a series of flattened sacs and consists of ribosomes on the cytosolic face. Ribosomes are the site of protein synthesis in cells. Rough ER manufactures membranes and secretory proteins, produces antibodies in certain leukocytes, and produces insulin in pancreatic cells. Other functions include initial N-linked glycosylation as assembly continues and manufacture of lysosomal enzymes.

It is a smooth tubule network and does not have ribosomes. It is usually interconnected with rough ER and serves as a transitional area for vesicles that transport ER products to various destinations. It has a wide range of functions which include lipid synthesis, carbohydrate metabolism, calcium concentration, drug detoxification, and attachment of receptors on cell membrane proteins. In muscles smooth ER assists in the contraction of muscle cells, and in brain cells it synthesizes male and female hormones.

An imbalance in the ER mediated protein folding can cause ER stress. The stress signaling pathway or the stress response of endoplasmic reticulum is known as the unfolded protein response (UPR). Initially UPR attempts to restore normal function of the cell by stopping protein translation and activate the signaling pathways that lead to increasing the production of molecular chaperones involved in protein folding. In case UPR fails in its initial attempts and the disruption prolongs, the UPR attempts to induce apoptosis.

Inhibitors mentioned below in Table 3 are used for inhibiting ER functions or induce ER stress, whereas activators mentioned in Table 4 are used to induce ER functions or counteract ER stress.

The Golgi apparatus is an organelle found in eukaryotic cells, and is a part of cell's endomembrane system. The Golgi apparatus is about 1 µM long, and consists of two components: flattened membranous sacs called cisternae and small membrane enclosed vesicles. It plays an important role for processing various proteins before secretion. Table 5 lists commonly used inhibitors of the Golgi apparatus.

The various functions of Golgi include: It receives proteins from protein containing vesicles from the rough endoplasmic reticulum, and further modifies them. It modifies, concentrates, sorts and packages the various macromolecules synthesized by the cell, before secretion or before they are sent to their cellular destinations. It modifies proteins by the addition of carbohydrates and phosphates. It plays a role in synthesizing proteoglycans belonging to the extracellular matrix of animals. Site for synthesis of polysaccharides present in the plant cell wall.

Secretion refers to the production and release of a useful substance by cells or a gland, including hormones, enzymes, cytokines and ECM proteins. The process by which materials are packaged in vesicles, and are secreted from a cell is known as exocytosis. Tables 6 and 7 list commonly used inhibitors and activators of secretion.

In eukaryotic cells, the classical process of secretion occurs through ER, Golgi and other intracellular vesicles, in a highly regulated process. Broadly, proteins targeted for outside dock and translocate through ER, after being synthesized by ribosomes. Vesicles containing the properly folded proteins then enter the Golgi apparatus. After glycosylation and other post-translational modifications, the proteins are moved into secretory vesicles which travel along the cytoskeleton to the edge of the cell. In the last step, exocytosis occurs where the vesicle fuses with cell membrane at a structure called the porosome and the proteins are released in the environment.

There are many proteins that are not secreted through the classical pathway involving ER and Golgi, and instead employ various pathways for secretion. These include FGF-1 (aFGF), FGF-2 (bFGF) and interleukin-1 (IL1) [4, 5]. The mechanisms can be broadly classified as two types. 1) Direct translocation of the substance across plasma membranes of cytoplasmic proteins e.g. FGF2 secretion. 2) Intracellular transport intermediates e.g acyl-CoA binding protein secretion [6].

Nuclear transport mainly occurs via the nuclear pore complexes (NPCs). Ions and small molecules are transported by passive diffusion through the NPCs. The import of larger nuclear proteins, RNAs, and ribonucleoproteins is mediated by nuclear localization signals (NLS) which mediate the docking at the nuclear pore. On the other hand, nuclear export is mediated by nuclear export signals (NES).

Nuclear import pathway broadly consists of four steps: 1) trimeric complex formation between the import substrate, importin α and importin β, 2) docking of the complex to NPC (Nuclear Pore Complexes), 3) translocation through the central channel, 4) dissociation of the complex and the release of the import substrate into the nucleoplasm. Table 8 lists commonly used inhibitors of nuclear transport.

Commonly peptides designed as NLS are conjugated to import substrate, to induce nuclear import. On the other hand, cell-permeable-peptides designed specific to nuclear transport receptors such as importins and transportins, which recognise NLS, are used for inhibiting nuclear transport [7].

Phospholipase C (PLC), Protein Kinase C (PKC), Rho GTPase, Rho kinase (downstream effector of RhoA), myosin light chain kinase (MLCK), and myosin light chain phosphatase (MLCPh) participate in the process of cell contraction at a particular intracellular Ca²⁺ concentration.

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Figure 2. Cell contraction.

Role of MLCK and MLCPh: Actin and myosin interactions are primarily responsible for mediating cell contraction. Phosphorylation of myosin light chain initiates the molecular interaction between actin and myosin. This phosphorylation of myosin light chain is determined by the balance between myosin light chain kinase (MLCK) activation and myosin light chain phosphatase (MLCPh).

Role of Rho GTPase and Rho-Kinase: Rho GTPase is activated in response to cell and microenvironment interactions through adhesion sites. Activated Rho GTPase binds to kinases such as serine/threonine kinases, Rho Kinase, ROK and the related p160ROCK; and elevate their activity. Activated Rho-Kinase inhibits myosin phosphatase activity leading to an increase in phosphorylated myosin light chain (MLC).

Role of PLC, Ca²⁺and PKC [8] : The process of contraction starts with activation of phospholipase C (PLC), which results in the production two second messengers namely diacylglycerol (DG) and inositol 1,4,5-trisphospate (IP3). IP3 causes the release of Ca²⁺ from the sarcoplasmic reticulum. Ca²⁺ can induce contraction in two ways: 1) Ca²⁺ along with DG activates protein kinase C. PKC further promotes contraction by phosphorylation of L-type Ca²⁺ channels and other proteins that regulate cross-bridge cycling, 2) Ca²⁺ binds to calmodulin and causes activation of myosin light chain kinase (MLCK).

Inhibitors and activators used to study cell contractility pathway are listed in Table 9 and Table 10.

Table 11 and Table 12 list commonly used inhibitors and activators of cell adhesion and cell junctions.

Cells use cell adhesion molecules to interact and adhere to a surface such as an extracellular matrix, other cells, or cell culture surface. Cell adhesion molecules (CAMs) include selectins, integrins and cadherins. Each type of adhesion molecule recognizes different molecules and has varied functions. Selectins are single chain transmembrane glycoproteins that bind to sugar moieties. There are 3 types of selectins namely L-, E-, and P-selectin [9-12]. Another important group is the Immunoglobulin Superfamily (IgSF CAMs). This family includes NCAMs (Neural Cell Adhesion Molecules), ICAM-1 (Intercellular Cell Adhesion Molecule), VCAM-1 (Vascular Cell Adhesion Molecule), and PECAM-1 (Platelet-endothelial Cell Adhesion Molecule).

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Figure 3. Cell adhesion molecules (CAMs). CAMs mostly belong to four families: integrins, cadherins, selectins and Ig superfamily which includes PECAM-1 and ICAM-1. CAMs participate either in homophilic interactions i.e. interactions with other CAMs of the same type, or in heterophilic interactions which are interactions with other CAMs or the extracellular matrix (ECM).

Cell-cell adhesions are mainly mediated by cadherins, a class of type-1 transmembrane proteins that are dependent on calcium ions for their function. They are a superfamily of cell adhesion molecules (CAMs) and are divided into subclasses, including E-, N- and P-cadherins [13-16].

Cell adhesion to ECM (extra cellular matrix) is mainly mediated by integrins. Integrins are a family of heterodimeric transmembrane glycoproteins. They are formed of large 'α' subunits of sized 120-170 kDa and small 'β' units of sizes 90-100 kDa. A variety of integrins are formed from 9 types of β subunits and 24 types of α subunits [17-20].

There are several types of cell junctions as discussed below. Synapses and neuromuscular junctions, part of the nervous systems, are to be considered as types of cell junction; they are not discussed here.

They include adherens junctions, desmosomes, and hemidesmosomes. Adheren junctions are observed in many cell types and are common in epithelial cells. The cytoplasmic face of the cell junction is liked to actin cytoskeleton. Here cadherin receptors bridge the neighboring plasma membranes via their homophilic interactions. Desmosomes are specialized for cell-cell adhesion, and are common in cells derived from the ectodermal lineages. They help cells in resisting mechanical stress. They are required in the epithelium, binding cells in muscle tissues, and maintaining the integrity of organs such as the skin and heart [21, 22]. Hemidesmosomes are similar in form to desmosomes. Unlike desmosomes which bind two cells together, hemidesmosomes bind one cell to the extracellular matrix.

They are composed of arrays of small channels where each channel is composed of two connexons, and directly link the cytoplasm of two cells. They facilitate the transport of various molecules and ions between two cells, thereby mediating electrical and mechanical coupling.

Also known as occluding junctions, seal the gap between two cells by forming an impermeable barrier. They directly link the cytoskeleton of two cells.

Frottin F et al treated HEK293T cells with 100 ng/ml of the RNA polymerase I inhibitor actinomycin D from MilliporeSigma to induce nucleolar disassembly in order to study nucleolus as a phase-separated protein quality control entity [23].

Labome surveys literature for the applications for antibodies and other reagents, and instruments. Table 13 lists activators and inhibitors cited in these articles, and their main suppliers.