Exocytosis – Definition, Steps, Types, Functions and Examples

Exocytosis is a cellular process by which materials are transported from inside of a cell to the extracellular region. In this process, membrane-bound vesicles fuse with the plasma membrane and release their contents outside the cell.

The process is as follows-

Vesicle formation → Vesicle transport → Tethering and docking → Fusion with plasma membrane → Release of vesicle contents

Exocytosis is used for the secretion of different substances such as hormones, neurotransmitters, enzymes and extracellular proteins. It has an important role in cell signaling, because the released molecules can act on nearby or distant cells. The vesicle membrane also becomes a part of plasma membrane. Thus, membrane proteins and lipids are delivered to cell surface and the plasma membrane is maintained.

In some cells, exocytosis is involved in removal of undigested or accumulated materials. This mainly occurs through lysosomal exocytosis, where a lysosome moves towards cell surface and fuses with the plasma membrane. It can also take part in plasma membrane repair and cellular clearance.

There are two main pathways of exocytosis. These are constitutive exocytosis and signal-regulated exocytosis. Constitutive exocytosis takes place continuously and does not require a special external signal. It supplies newly formed proteins and lipids to the plasma membrane and extracellular region.

Signal-regulated exocytosis occurs only after a specific stimulus is received. The vesicles remain stored inside the cell before release. A rise in intracellular Ca²⁺ is a common signal, especially during the secretion of neurotransmitters, hormones and digestive enzymes.

What Is Exocytosis?

Exocytosis is a type of vesicular transport by which substances are moved from inside of the cell towards the cell surface. The substances are generally carried in membrane-bound vesicles.

The vesicles move through the cytoplasm and reach the plasma membrane. The membrane of vesicle then fuses with the plasma membrane. By this fusion, the inside of vesicle becomes connected with the outside of cell.

The soluble materials present within vesicle are released into the extracellular space. These materials may include proteins, hormones, enzymes, neurotransmitters or other secretory substances, depending on the type of cell.

During the process, the membrane of vesicle is also added to the plasma membrane. The vesicular lipids become a part of the lipid bilayer. Membrane proteins carried by the vesicle are also incorporated into the plasma membrane.

Exocytosis is classified as a form of vesicular transport. It is also included under bulk transport, because the materials are carried with the help of vesicles instead of passing directly through the plasma membrane.

Main Characteristics of Exocytosis

The following are the main characteristics of exocytosis–

• Exocytosis is a type of vesicular transport or bulk transport. The materials are carried within the membrane-bound vesicles and not passed directly through the lipid bilayer.
• It is directed from the interior of cell towards the cell surface. The vesicle first moves to its particular target region of the plasma membrane.
• During this process, the vesicle is tethered and then docked with the plasma membrane. In regulated secretion an ATP-dependent priming step is also present before membrane fusion.
• The membrane of vesicle fuses with the plasma membrane. A small fusion pore is formed, which connects the vesicle lumen with extracellular region.
• Soluble substances present inside the vesicle are released through the fusion pore. These may be neurotransmitters, hormones, enzymes and different secretory proteins.
• The fusion pore may enlarge and the vesicle completely joins with plasma membrane. In some exocytic events, the pore opens for a short period and then closes. This is referred to as kiss-and-run exocytosis.
• The lipids of vesicular membrane become added to the plasma membrane. Its membrane proteins are also delivered to cell surface, including receptors, channels or transport proteins.
• Exocytosis has both constitutive and regulated forms. Constitutive exocytosis takes place continuously for membrane and protein delivery. Regulated exocytosis takes place after receiving a particular signal.
• An increase in intracellular Ca²⁺ is a common stimulus for regulated exocytosis. It is particularly present during neurotransmitter and hormone release. All exocytosis, however, is not controlled by Ca²⁺.
• Different proteins control vesicle recognition and fusion. Rab proteins, tethering factors and SNARE proteins help the vesicle to reach and fuse with its correct membrane.
• Several steps of exocytosis require cellular energy. Vesicle movement and priming use ATP or GTP-dependent mechanisms, whereas the final membrane fusion is mainly driven by assembly of the fusion machinery.
• Exocytosis increases the plasma membrane surface when the vesicle membrane is added. The excess membrane can later be recovered through endocytosis, which helps in maintaining membrane balance.
• It is used for secretion, cell-to-cell signaling, insertion of membrane components, plasma membrane repair and removal of some cellular materials. Thus, exocytosis is not limited only to the release of secretory products.

Cellular Components Involved in Exocytosis

Different membrane structures and proteins are involved in exocytosis. These components help in vesicle formation, its movement, docking and final fusion. The major cellular components are as follows-

1. Golgi Apparatus

The Golgi apparatus receives proteins and lipids from the endoplasmic reticulum. It modifies and sorts these materials. At the trans-Golgi network, many secretory cargos are packed into transport vesicles or secretory granules.

2. Secretory Vesicles

Secretory vesicles are small membrane-bound sacs which carry the substances for exocytosis. Their cargo can be soluble proteins, enzymes, hormones or neurotransmitters. Some vesicles also carry receptors, channels and other membrane proteins.

In constitutive exocytosis, the vesicles are transported and fused continuously. The regulated secretory vesicles remain stored inside the cell until a particular signal is received.

3. Cytoskeleton

The cytoskeleton forms the transport path for movement of vesicles. Microtubules are generally used for movement over longer distance. Actin filaments have more role near the cell cortex and plasma membrane.

Actin can help the vesicle to reach its release site. It may also form a barrier, depending on the cell and stage of secretion.

4. Motor Proteins

Motor proteins move the vesicles along the cytoskeletal fibres. Kinesin and dynein are associated with microtubule-based movement. Different myosin proteins carry or position vesicles on actin filaments.

These proteins use energy from ATP. Thus, the vesicle is transported towards its particular region of cell surface.

5. Rab GTPases

Rab proteins are small GTP-binding proteins associated with vesicular membranes. They help in identification of the vesicle and its correct target membrane.

Active Rab proteins bind with different effector proteins. These effectors are used for vesicle movement, tethering and docking. Rab3 and Rab27 have important functions in several forms of regulated secretion.

6. Tethering Proteins

Tethering proteins first capture the vesicle close to the plasma membrane. This takes place before tight docking and membrane fusion.

The exocyst complex is an important vesicle-tethering complex. It is formed of eight protein subunits and is used in the delivery of several exocytic vesicles to particular regions of plasma membrane.

7. SNARE Proteins

SNARE proteins are the major membrane fusion proteins of exocytosis. A vesicular SNARE binds with its corresponding SNARE proteins on the target membrane.

VAMP or synaptobrevin is commonly present on the vesicle membrane. Syntaxin and SNAP-25 or related proteins are present on the plasma membrane. Their assembly pulls the two membranes close and promotes membrane fusion. The exact SNARE type is different among cells and exocytic pathways.

8. Priming and SNARE-Regulating Proteins

Docked vesicles are made ready for fusion by a process called priming. This step is ATP-dependent in many regulated secretory pathways.

Munc13 proteins take part in vesicle priming. Munc18, an SM protein, binds with syntaxin and controls the formation and functioning of SNARE complex. These proteins are especially well studied in synaptic and neuroendocrine exocytosis.

9. Calcium Ions and Synaptotagmin

An increase in intracellular Ca²⁺ acts as the signal for many forms of regulated exocytosis. Calcium enters through membrane channels or may be released from an intracellular store.

Synaptotagmin is a vesicular calcium-binding protein. In fast neurotransmitter release, it acts as an important Ca²⁺ sensor. After binding calcium, it interacts with phospholipids and the fusion machinery, which triggers rapid vesicle fusion. It is not the calcium sensor for every type of exocytosis.

10. Plasma Membrane

The plasma membrane is the target membrane during exocytosis. It contains target SNAREs, phospholipids and other proteins required for docking and fusion.

The vesicle membrane joins with this membrane. Soluble cargo is then released outside of the cell, while vesicular lipids and membrane proteins become added into the plasma membrane.

11. NSF and SNAP Proteins

After complete fusion, the assembled SNARE complex has to be separated. NSF and soluble NSF attachment proteins (SNAPs) are involved in this step.

NSF uses energy from ATP for disassembly of the SNARE complex. The separated SNARE proteins can then be used again for another vesicle transport and fusion event.

Stages of Exocytosis

Exocytosis takes place by a sequence of vesicle movement and membrane-fusion events. The stages are not completely same in every cell. Priming and signal activation are mainly important in regulated exocytosis.

The following are the stages of exocytosis-

1. Vesicle Formation and Cargo Packaging

In the first step, secretory materials are packed within a membrane-bound vesicle. Many exocytic vesicles are formed from the trans-Golgi network (TGN) after sorting of proteins and lipids.

The cargo present within vesicle may be soluble or membrane-bound. Soluble cargo remains in its lumen, whereas membrane proteins are placed within vesicular membrane.

2. Vesicle Transport

The newly formed vesicle is moved from the cell interior towards the plasma membrane. Microtubules and actin filaments are used during this movement.

Motor proteins carry the vesicles along these cytoskeletal tracks. Movement over longer distance generally takes place on microtubules. Near the cell surface, actin-based movement may occur.

3. Vesicle Tethering

In this step, the vesicle is first captured near its particular region of the plasma membrane. It is not tightly attached at this stage.

Rab GTPases and tethering factors help in recognition of the correct target membrane. Thus, a secretory vesicle does not normally fuse with any random cellular membrane.

4. Vesicle Docking

After tethering, vesicle comes into close contact with plasma membrane. This close attachment is referred to as docking.

The proteins of vesicle and target membrane now interact with each other. Docked vesicles remain at or very close to the site where fusion will take place.

5. Vesicle Priming

The docked vesicle is then prepared for membrane fusion. This step is called priming.

During priming, the fusion proteins become arranged into a fusion-ready condition. It is an ATP-dependent step in many regulated secretory cells. Priming is especially present in neurons, endocrine cells and other cells containing stored secretory vesicles.

6. Signal Activation

In regulated exocytosis, the primed vesicle remains stored until a particular signal is received. An increase in cytoplasmic Ca²⁺ is the common signal in many cells.

Calcium binds with calcium-sensing proteins such as synaptotagmin in fast neuronal secretion. This activates the final fusion reaction. Constitutive exocytosis does not generally wait for this acute Ca²⁺ signal.

7. Membrane Fusion

The membrane of vesicle is pulled close to the plasma membrane. The SNARE proteins have a major role during this process.

The vesicle and plasma membrane lipid bilayers then merge. A narrow connection is formed between the vesicle lumen and extracellular region. This is called the fusion pore.

8. Cargo Release

The fusion pore allows soluble vesicular cargo to pass into the extracellular space. The pore may become wider, so the materials are released outside of cell.

In full-fusion exocytosis, the vesicle completely collapses into plasma membrane. In another condition, the pore opens temporarily and again closes. This is referred to as kiss-and-run exocytosis.

9. Addition of Vesicle Membrane

After fusion, vesicular lipids become added to the plasma membrane. The membrane proteins carried by vesicle are also exposed at the cell surface.

Thus, exocytosis not only releases soluble cargo. It also supplies new lipids, receptors, channels and other membrane proteins to the plasma membrane.

10. Membrane Recovery

The membrane added during exocytosis may later be recovered by endocytosis. This prevents a continuous unwanted increase in plasma membrane area.

Recovered membrane can be recycled for formation of new vesicles. This is a post-exocytic event and is closely connected with the exocytosis cycle.

Molecular Mechanism of Vesicle Fusion

Vesicle fusion is the final process where the membrane of secretory vesicle joins with the plasma membrane. It is mainly carried out by SNARE proteins with Rab proteins, tethering factors and different regulatory proteins.

The following are the step by step molecular mechanism of vesicle fusion-

1. Recognition of Target Membrane

In the first step, the secretory vesicle reaches near the particular region of plasma membrane. Rab GTPase is present in its active GTP-bound form on vesicular membrane.

Rab binds with its particular effector or tethering protein. It helps in recognition of correct target membrane. Thus, the vesicle is not allowed to fuse with any other cellular membrane.

2. Tethering of Vesicle

After recognition, the vesicle is loosely captured by the tethering proteins. This first attachment is called tethering.

The tethering factors hold both membranes near to each other. They also help in the arrangement of SNARE proteins for next process. Different exocytic pathways contain different types of tethering complex.

3. Docking and Priming

The tethered vesicle now comes into close contact with plasma membrane. This is referred to as docking.

In regulated exocytosis, the docked vesicle is made fusion ready by priming. Munc18 binds with the plasma-membrane SNARE called syntaxin. Munc13 helps to open syntaxin and allows formation of the SNARE complex.

4. Formation of trans-SNARE Complex

The vesicle contains an R-SNARE, commonly called vesicular SNARE or v-SNARE. In neuronal vesicles, it is VAMP2 or synaptobrevin.

The plasma membrane contains Q-SNAREs or target SNAREs. Syntaxin-1 and SNAP-25 are the common neuronal examples. The SNARE types, however, are different in other secretory pathways.

The vesicular and target SNAREs bind with each other across the two membranes. This forms a trans-SNARE complex.

5. SNARE Zippering

Four SNARE helices assemble into a tight helical bundle. The complex starts its assembly from N-terminal region and moves towards the membrane-attached C-terminal region. This process is called SNARE zippering.

During this process, the vesicle membrane and plasma membrane are pulled very close. The energy released by formation of SNARE complex is used to overcome the energy barrier between the two lipid bilayers.

6. Calcium Triggering in Regulated Exocytosis

In many regulated secretory cells, the partly assembled SNARE complex remains in a fusion-ready state. Complexin and other regulatory proteins control premature membrane fusion.

After stimulation, intracellular Ca²⁺ concentration increases. Calcium binds with synaptotagmin, particularly synaptotagmin-1 during fast neurotransmitter release. It then interacts with phospholipids and the SNARE machinery, which helps in triggering the final fusion. This calcium step is not required in the same form for all exocytic pathways.

7. Lipid Bilayer Rearrangement

The two membranes are now placed at a very small distance. Water molecules present between their lipid surfaces are displaced and the membrane lipids become disturbed.

The outer leaflets of the two lipid bilayers may first join and form a hemifusion intermediate. The inner leaflets remain separate for a short condition. The exact intermediate can differ and some details of this step are still under study.

8. Formation of Fusion Pore

After further membrane rearrangement, a small aqueous opening is formed. This is called the fusion pore.

The fusion pore connects the lumen of vesicle with the extracellular region. Soluble cargo now starts to pass through the pore and is released outside of the cell.

9. Fusion Pore Expansion

The initial fusion pore may expand and the vesicle membrane completely collapses into the plasma membrane. This is called full-collapse fusion.

In some conditions, the pore remains open for a short time and again closes. The vesicle then separates without complete collapse. This is referred to as kiss-and-run fusion.

10. Formation of cis-SNARE Complex

After complete fusion, all SNARE proteins become present within the same plasma membrane. This post-fusion complex is called the cis-SNARE complex.

The vesicular lipids and membrane proteins are also incorporated into plasma membrane. Soluble vesicular cargo has already been released outside.

11. Disassembly and Recycling of SNAREs

The cis-SNARE complex is very stable and cannot easily separate by itself. α-SNAP binds with this complex and recruits the ATPase protein called NSF.

NSF hydrolyses ATP and separates the SNARE proteins. These proteins can be sorted and again used during another vesicle-fusion process.

Types of Exocytosis

Based on whether vesicle fusion takes place continuously or after receiving a signal, exocytosis can be classified into two main types:

1. Constitutive Exocytosis
2. Regulated Exocytosis

The process can also be divided based on the way by which vesicle membrane fuses with the plasma membrane.

1. Constitutive Exocytosis

It is a type of exocytosis where the vesicles continuously fuse with the plasma membrane. No particular external signal is needed for each fusion event.

The vesicles are generally formed from the trans-Golgi network (TGN). After reaching the cell surface, they fuse and release their soluble materials outside of cell.

Constitutive exocytosis also adds newly formed lipids and membrane proteins to the plasma membrane. It is present in all eukaryotic cells and is used for normal membrane growth, replacement and continuous secretion.

Some examples are the secretion of extracellular matrix proteins, antibodies and different proteins that are released continuously.

Constitutive exocytosis is a continuous pathway for transport of proteins and lipids towards the cell surface. The vesicles do not remain stored for receiving a particular acute signal.

The following are the steps of constitutive exocytosis-

1. In the first step, secretory proteins and membrane proteins are synthesized in the rough endoplasmic reticulum (RER). The newly formed proteins enter into ER lumen or become inserted into its membrane.
2. The proteins are packed into COPII-coated transport carriers at the ER exit sites. These carriers move from ER towards the Golgi apparatus.
3. The cargo enters into the cis face of Golgi apparatus. It passes through the Golgi cisternae where different modification and processing takes place.
4. At the trans-Golgi network (TGN), proteins and lipids are sorted according to their destination. Cargo intended for secretion or plasma membrane is packed into post-Golgi transport carriers.
5. The constitutive carriers leave the TGN soon after their formation. They are not stored as mature secretory granules. A particular calcium signal is also not required for each vesicle release.
6. The post-Golgi carriers move through the cytoplasm. Microtubules, actin filaments and motor proteins can take part during this movement. The carrier reaches towards its particular region of plasma membrane.
7. Rab GTPases and tethering proteins help in recognition of the target membrane. The exocyst complex can capture the secretory carrier near the plasma membrane. This step is referred to as tethering.
8. The vesicle now comes into close contact with plasma membrane. Vesicular SNARE protein binds with its corresponding target-membrane SNARE proteins. The two membranes are brought close during this process.
9. The vesicle membrane fuses with the plasma membrane. A fusion pore is formed and the inside of vesicle becomes connected with extracellular region.
10. Soluble cargo is released outside of cell. The lipids and membrane proteins of vesicle are incorporated into the plasma membrane.
11. The SNARE complex is separated after fusion and its proteins can be used again. Some of the membrane added by exocytosis is later recovered through endocytosis. It helps in maintaining the plasma-membrane area.

Constitutive exocytosis does not mean that the pathway is completely without regulation. Its rate can be changed by cellular signals and membrane receptors. The vesicles, however, are not kept in a stored pool for signal-triggered release.

2. Regulated Exocytosis

Regulated exocytosis is a type where the secretory vesicles remain stored within the cytoplasm. They do not fuse immediately after reaching near the plasma membrane.

Fusion takes place when the cell receives a particular stimulus. The stimulus commonly increases the level of intracellular Ca²⁺, but the exact signal is different according to cell type.

It is mainly found in cells which release their products rapidly when required. For example, neurons release neurotransmitters, endocrine cells release hormones and exocrine cells release digestive enzymes through this pathway.

The amount and timing of release are controlled in this type. The vesicle may remain docked and primed before the signal is received.

The following are the steps of regulated exocytosis-

1. Secretory proteins are sorted at the trans-Golgi network (TGN). These materials are packed within immature secretory vesicles or secretory granules.
2. The immature vesicle undergoes maturation. The vesicle lumen becomes acidic and some cargo proteins are processed. Secretory substances may also become condensed.
3. Mature secretory vesicles remain stored within the cytoplasm. Some vesicles are located near plasma membrane, while others remain in reserve pool.
4. The vesicles move towards the cell surface with the help of microtubules, actin filaments and motor proteins.
5. Rab proteins and tethering proteins help the vesicle to reach its correct region of plasma membrane. The vesicle is then tethered near the fusion site.
6. The vesicle comes into close contact with plasma membrane. This step is called docking.
7. The docked vesicle is prepared for membrane fusion. This process is referred to as priming. Munc13, Munc18 and other regulatory proteins take part in it.
8. A particular stimulus is received by the cell. It may be an action potential, hormone, antigen or another cellular signal.
9. The stimulus generally increases the cytoplasmic Ca²⁺ level. Calcium enters from outside of cell or it is released from an intracellular calcium store.
10. Calcium binds with a calcium-sensing protein. Synaptotagmin acts as an important calcium sensor in many regulated secretory pathways.
11. The vesicular SNARE protein binds with SNARE proteins present on plasma membrane. These proteins form the SNARE complex and bring the two membranes close.
12. The vesicle membrane fuses with plasma membrane. A small fusion pore is formed between the vesicle lumen and extracellular space.
13. Secretory materials are released outside of cell through the fusion pore. The pore may become wider and the vesicle completely joins with plasma membrane.
14. In some conditions, the fusion pore opens for a short time and then closes. The vesicle separates without complete collapse. This is referred to as kiss-and-run exocytosis.
15. After complete fusion, the vesicle membrane becomes added to plasma membrane. Its lipids and membrane proteins also become a part of cell surface.
16. The added membrane is later recovered by endocytosis. The SNARE proteins are also separated and used again for another vesicle-fusion process.

Types Based on Mode of Vesicle Fusion

Based on the behaviour of fusion pore and vesicle membrane, exocytosis has the following modes-

3. Full-Collapse Fusion

In full-collapse fusion, the fusion pore is formed and then becomes wider. The complete vesicle membrane collapses into the plasma membrane.

Most or all soluble cargo is released into the extracellular region. The vesicular lipids and proteins become a part of plasma membrane. Membrane is later recovered by endocytosis.

4. Kiss-and-Run Exocytosis

In this type, the vesicle forms a temporary fusion pore with plasma membrane. The pore remains open for a short period and some or all releasable cargo passes through it.

The fusion pore then closes. Vesicle separates from the plasma membrane without complete collapse. This is referred to as kiss-and-run exocytosis.

It is commonly studied in synaptic and neuroendocrine vesicles. The vesicle membrane can be reused more rapidly in this process.

5. Compound Exocytosis

Compound exocytosis involves the fusion of two or more vesicles before or during secretion. One vesicle may fuse with plasma membrane and other vesicles then fuse with this vesicle.

A larger vesicular structure is formed. This allows release of a large quantity of secretory material during strong stimulation.

It is present in several secretory cells, including mast cells and some endocrine or exocrine cells. Compound exocytosis is considered one of the major exocytic fusion modes.

Specialized Forms of Exocytosis

6. Lysosomal Exocytosis

Lysosome-mediated exocytosis is a type of regulated exocytosis where a lysosome fuses with the plasma membrane. The lysosomal contents are released outside of cell and its membrane becomes added to the plasma membrane.

Lysosomes are generally involved in degradation of cellular materials. But they can also function as Ca²⁺-regulated secretory organelles. This process has been found in fibroblasts, epithelial cells, muscle cells and several other cell types.

The following are the steps of lysosome-mediated exocytosis-

1. The lysosome moves from inner region of cell towards the plasma membrane. Microtubules and motor proteins help during this movement.
2. A rise in cytoplasmic Ca²⁺ acts as the major signal. Calcium may enter through damaged plasma membrane or released from intracellular calcium stores.
3. The lysosome reaches near the plasma membrane. It is then tethered and docked at the particular fusion site.
4. VAMP7 present on lysosomal membrane binds with syntaxin-4 and SNAP-23 present on plasma membrane.
5. These proteins form the SNARE complex. It brings lysosomal membrane and plasma membrane very close.
6. Synaptotagmin VII binds with calcium. It helps in activation of the final membrane-fusion process.
7. The lysosomal membrane fuses with plasma membrane. A small fusion pore is formed between lysosome and extracellular region.
8. The fusion pore becomes wider. Lysosomal enzymes and other soluble materials are released outside of cell.
9. The lysosomal membrane becomes added to plasma membrane. The luminal region of LAMP1 is now exposed on outer surface of cell.
10. The released lysosomal substances take part in plasma-membrane repair, extracellular degradation or removal of accumulated materials.

7. Non-Secretory Exocytosis

All exocytic vesicles do not carry soluble materials for secretion. Some vesicles mainly deliver membrane lipids, receptors, channels, pumps or transport proteins to the cell surface.

This is called non-secretory exocytosis. It is used during membrane expansion, cell migration, cytokinesis, plasma membrane repair and regulation of proteins present at cell surface.

Constitutive versus Regulated Exocytosis

Constitutive exocytosis and regulated exocytosis are two major pathways by which vesicles fuse with the plasma membrane. The major difference is in their storage, signal requirement and time of release.

The differences between constitutive and regulated exocytosis are as follows-

Both pathways use vesicle targeting and membrane-fusion machinery. Rab proteins, tethering factors and SNARE proteins can take part in them. The difference is mainly how the vesicle is stored and when its fusion is allowed.

What Triggers and Regulates the Exocytosis Process?

Exocytosis is regulated by cellular signals and different proteins present on vesicle and plasma membrane. In regulated exocytosis, vesicles remain stored inside the cell. The fusion starts after receiving a particular signal. The exact signal is not same in all cells.

The major factors triggering and regulating exocytosis are-

1. Calcium ions (Ca²⁺)– It is the most common trigger of regulated exocytosis. After stimulation, calcium enters into the cell or it is released from intracellular calcium stores. The rise in calcium level starts fusion of the primed vesicles. Every exocytic process, however, is not triggered by calcium.
2. Membrane depolarization– It is mainly involved in nerve cells and several endocrine cells. Membrane depolarization opens voltage-gated calcium channels. Calcium enters near the docked vesicle and the secretory product is released.
3. Extracellular signals– Hormones, neurotransmitters, antigens or other signaling molecules can bind with the cell surface receptors. This binding activates intracellular signaling pathway. The signal finally reaches the secretory vesicles and controls their fusion.
4. Synaptotagmin– It is a calcium-binding protein associated with several secretory vesicles. After binding with Ca²⁺, synaptotagmin interacts with the membrane phospholipids and fusion proteins. It acts as an important calcium sensor during rapid neurotransmitter release. Different synaptotagmins are present in other secretory pathways.
5. SNARE proteins– These are the major proteins used during vesicle fusion. The SNARE of vesicle binds with the SNARE proteins present on plasma membrane. This binding brings the two membranes close.

In neurons, VAMP2 (synaptobrevin-2) is present on vesicle, whereas syntaxin-1 and SNAP-25 are present on plasma membrane. Their complex provides the force required for fusion.

6. Rab GTPases– Rab proteins are used for movement and correct targeting of vesicles. The active GTP-bound Rab binds with its effector proteins. It helps the vesicle to reach its particular fusion site. Rab3 and Rab27 are involved in many regulated secretory processes.
7. Tethering proteins– These proteins first capture the vesicle near plasma membrane. It prevents the fusion of vesicle with a wrong cellular membrane. After tethering, close docking is carried out.
8. Munc13 and Munc18 proteins– These proteins are involved in preparation of vesicle before fusion. This preparation is called priming. Munc13 and Munc18 help in proper formation of the SNARE complex. The primed vesicle can now respond rapidly after calcium signal.
9. Complexin– Complexin binds with the partly formed SNARE complex. It controls unsuitable fusion before calcium entry. During stimulation, it also takes part in rapid calcium-triggered release. Its exact effect is different according to cell and vesicle type.
10. Second messengers– Second messengers such as cAMP, IP₃ and DAG also regulate secretion. IP₃ can increase calcium release from an intracellular store. DAG activates protein kinase C (PKC).

cAMP acts through protein kinase A (PKA) or Epac proteins. These pathways can increase vesicle priming, calcium sensitivity or the amount of secretion. They are not the main trigger in every cell.

11. Cytoskeleton and motor proteins– Microtubules, actin filaments and motor proteins carry secretory vesicles towards the cell surface. Cortical actin may also prevent vesicle access to plasma membrane. During stimulation, its arrangement is changed and vesicles are allowed to move towards the fusion site.
12. Number of primed vesicles– All secretory vesicles are not ready for immediate fusion. Only docked and primed vesicles form the readily releasable pool. The size of this pool controls the amount of material released after stimulation.

Functions and Biological Importance of Exocytosis

Exocytosis is not only used for secretion of substances. It also supplies membrane lipids and proteins to the cell surface. Some of the important functions and biological importance of exocytosis are as follows-

• Secretion of neurotransmitters– Neurotransmitters stored within synaptic vesicles are released by exocytosis. It allows transmission of nerve signal from one neuron to another cell. The release takes place rapidly after calcium enters into the nerve terminal.
• Release of hormones– Peptide hormones are packed within secretory granules and released outside the endocrine cell. Insulin release from pancreatic β-cells is one of its common example. Thus, exocytosis has an important role in hormonal regulation of different body functions.
• Secretion of enzymes– Different secretory cells release enzymes with the help of exocytosis. The pancreatic acinar cells release digestive enzymes from zymogen granules after stimulation. These enzymes are required for digestion of food.
• Cell signaling– The substances released by exocytosis can act on nearby or distant cells. Neurotransmitters, hormones and other signaling proteins are included in it. In this way, exocytosis is used for communication between cells.
• Addition of membrane lipids and proteins– During vesicle fusion, the vesicular membrane becomes a part of the plasma membrane. Its lipids are added into lipid bilayer. Receptors, channels, transport proteins and other membrane proteins can also reach the cell surface by this process.
• Growth and maintenance of plasma membrane– New membrane is required during cell growth and formation of cellular extensions. Exocytic vesicles provide a major source of this membrane. The excess or old membrane is later recovered by endocytosis, which maintains the membrane balance.
• Cell polarity and development– Polarized exocytosis delivers proteins and lipids to particular regions of cell surface. It is important for formation of apical and basolateral membrane regions in epithelial cells. Exocytosis also takes part in junction formation, morphogen secretion and tissue development.
• Plasma membrane repair– Damage in plasma membrane causes entry of calcium into the cell. This can trigger fusion of lysosomes with the damaged membrane. The added lysosomal membrane helps in closing or removing the injured region.
• Extracellular matrix formation and remodelling– Extracellular proteins are transported outside of cell by the exocytic pathway. These materials help in formation of extracellular matrix. Lysosomal exocytosis can also release enzymes which take part in its remodelling.
• Immune defence– Cytotoxic T cells and natural killer cells release lytic granules by regulated exocytosis. These granules contain perforin and granzymes, which help in destruction of infected or transformed cells. Mast cells also release inflammatory mediators through granule exocytosis.
• Fertilization– Exocytosis is involved in the acrosome reaction of sperm. The acrosomal membrane fuses and its contents are released. This reaction is required for proper interaction of sperm with the coverings of egg and fertilization.
• Cell division– Exocytic vesicles are delivered near the site of separation of daughter cells. The vesicle membrane and proteins help during the final abscission step of cytokinesis. Disturbance of this transport can prevent complete separation of two cells.
• Cell movement and nerve growth– Local exocytosis adds new membrane towards a growing or moving part of cell. It is used during extension of axons and other cellular processes. The process also takes part during cellular wound healing.
• Removal of some cellular materials– Lysosomes can fuse with plasma membrane and release their contents outside. This process can take part in removal of some undigested or accumulated materials. It is not the general route for all cellular wastes.

Examples of Exocytosis in Different Cells

Exocytosis takes place in different secretory and non-secretory cells. The type of vesicle and the released material is different according to the function of cell.

Some of the important examples are as follows-

1. Exocytosis in Nerve Cells

An example of exocytosis is the release of neurotransmitters from a neuron. The neurotransmitters are stored within synaptic vesicles at the presynaptic terminal.

After arrival of nerve impulse, voltage-gated calcium channels are opened. Ca²⁺ enters into the terminal and the vesicles fuse with presynaptic membrane. The neurotransmitter is then released into the synaptic cleft.

2. Exocytosis in Pancreatic Beta Cells

Another example can be observed in the beta cells of pancreas. Insulin is stored within the dense-core secretory granules.

An increase in blood glucose stimulates the beta cells. The insulin granules move towards cell surface and fuse with the plasma membrane. Insulin is released into blood by regulated exocytosis.

3. Exocytosis in Pancreatic Acinar Cells

Pancreatic acinar cells produce different digestive enzymes. These enzymes are packed within membrane-bound zymogen granules.

After stimulation by acetylcholine or cholecystokinin, intracellular calcium level is increased. The zymogen granules fuse at the apical membrane and enzymes are released into the pancreatic duct.

4. Exocytosis in Mast Cells

Mast cells contain secretory granules having histamine, proteases and other inflammatory substances. During an allergic reaction, antigen can activate the IgE-bound receptors of mast cell.

The secretory granules then fuse with plasma membrane and their contents are released. Several granules may also fuse with one another during compound exocytosis, followed by a large discharge of granule materials.

5. Exocytosis in Cytotoxic T Cells and Natural Killer Cells

Cytotoxic T cells and natural killer (NK) cells contain secretory lysosomes called lytic granules. These granules have perforin and granzymes.

After recognition of a target cell, the granules are moved towards the immunological synapse. They fuse with the plasma membrane and release the lytic proteins near target cell. The released substances help in killing infected or abnormal cells.

6. Exocytosis in Goblet Cells

Goblet cells are secretory epithelial cells which produce mucins. The mucins are packed and stored within large secretory granules.

The granules are released by exocytosis at the apical surface. After release, mucins absorb water and form mucus. This mucus covers and protects the intestinal epithelial surface.

7. Exocytosis in Sperm Cells

The acrosome present in sperm head is a single large secretory vesicle. It contains different proteins required during fertilization.

During acrosomal exocytosis, the outer acrosomal membrane fuses at several places with the overlying plasma membrane. Acrosomal materials are released and the membrane arrangement of sperm head is changed. This process takes part during interaction of sperm with the coverings of egg.

8. Exocytosis in Plant Cells

Exocytosis also occurs in plant cells. Vesicles formed from Golgi apparatus carry cell-wall materials and membrane proteins towards the plasma membrane.

The vesicle membrane fuses with plasma membrane and the cargo is delivered outside or added into membrane. Cellulose synthase complexes are also inserted into the plasma membrane by this process. It is important during cell-wall formation and plant cell growth.

Methods Used to Study Exocytosis

Different electrical, optical and biochemical methods are used for studying exocytosis. Some methods detect the fusion of vesicle membrane. Other methods measure the substance released from vesicle.

Some of the important methods are as follows-

1. Electron Microscopy

Electron microscopy is used to observe secretory vesicles and their relationship with the plasma membrane. Docked vesicles, fused membrane profiles and fusion pores can be studied by this method.

Quick-freezing and freeze-fracture electron microscopy can arrest the cells during early exocytotic events. It gives a highly detailed structure, but continuous fusion in a living cell is not recorded.

2. Membrane Capacitance Measurement

The plasma membrane acts as an electrical capacitor. When a vesicle fuses, its membrane becomes added to plasma membrane and the membrane capacitance is increased.

The change is measured with the patch-clamp technique. Whole-cell recording is used for total exocytosis, whereas cell-attached recording can detect fusion of individual vesicles. It measures membrane addition but does not identify the substance released.

3. Carbon-Fibre Amperometry

Amperometry is used for detection of electroactive substances such as catecholamines and serotonin. A carbon-fibre microelectrode is placed close to the secreting cell.

The released molecule is oxidized at electrode and produces a current spike. Each spike can represent the release from a single secretory vesicle. The spike area is used to estimate amount of material released. It cannot directly detect a cargo which is not electroactive.

4. Patch Amperometry

Patch amperometry is a combined method of membrane capacitance measurement and amperometry. It detects vesicle fusion and release of its chemical contents at same time.

The capacitance signal shows addition of vesicle membrane. The amperometric signal detects the released transmitter. It is also used for studying opening and expansion of the fusion pore.

5. Total Internal Reflection Fluorescence Microscopy

Total internal reflection fluorescence microscopy (TIRFM) is used to observe fluorescent vesicles very close to the plasma membrane. Only a thin area near the glass-cell surface is illuminated.

The movement, docking and disappearance of a labelled vesicle can be recorded. Individual fusion events can also be counted. It is commonly used for secretory granules and vesicles present near the basal cell membrane.

6. pHluorin-Based Fluorescence Method

pHluorin is a pH-sensitive form of green fluorescent protein. It is joined with a vesicular membrane protein in such a way that the fluorescent part remains inside vesicle.

Its fluorescence is low in the acidic vesicle lumen. During exocytosis, it is exposed to the nearly neutral extracellular fluid and a bright fluorescent signal is formed. After endocytosis and vesicle acidification, the signal again decreases. This method is mainly used for real-time study of synaptic-vesicle cycling.

7. FM Dye Method

FM1-43 and related styryl dyes bind with the outer leaflet of plasma membrane. During endocytosis, some dye becomes trapped within the recycled synaptic vesicles.

When the labelled vesicles undergo exocytosis, dye is released into surrounding medium and fluorescence is decreased. This loss of fluorescence is called destaining. FM dyes are used to study vesicle pools, exocytosis and recycling at nerve terminals.

8. Measurement of Secreted Products

The substance released into culture medium can be measured after stimulation of cells. Hormones, cytokines, enzymes and other secretory products are detected by ELISA, radioimmunoassay, enzyme-activity assay or other biochemical method.

Static incubation gives the total secretion during a selected period. A perifusion system collects samples continuously and is used for studying the time course of secretion. These methods measure released material from many cells, but do not directly show each vesicle-fusion event.

9. Flow Cytometry and Surface-Marker Method

Some vesicular proteins have a luminal region which is normally present inside the vesicle. After fusion, this region becomes exposed on the outer cell surface.

Fluorescent antibodies against LAMP-1 (CD107a), LAMP-2 or CD63 can be used for its detection. The signal is measured by flow cytometry or fluorescence microscopy. This method is commonly used for lysosomal exocytosis and degranulation of immune cells.

10. Calcium Imaging

Regulated exocytosis is commonly associated with an increase of intracellular Ca²⁺. Fluorescent calcium indicators are used to measure the calcium change before and during secretion.

Calcium imaging can be combined with amperometry or vesicle fluorescence. In this way, calcium entry and exocytotic release can be compared in the same cell. A calcium rise alone, however, does not confirm that vesicle fusion has occurred.

11. Genetic and Pharmacological Methods

Genes coding for SNARE proteins, Rab proteins, synaptotagmins and other exocytotic components can be knocked down, removed or mutated. Exocytosis is then measured by capacitance, fluorescence or secretion assay.

Toxins and chemical inhibitors are also used for blocking a selected step. The change produced after treatment helps in finding the function of that protein. These methods do not directly detect exocytosis and are generally used together with another measuring method.

Endocytosis and Exocytosis Compared

Endocytosis and exocytosis are two processes of vesicular transport. Endocytosis carries substances into the cell, whereas exocytosis carries vesicular materials towards cell surface or outside of cell. These act in opposite direction but both are connected with cellular membrane trafficking.

Major Differences

The major differences between endocytosis and exocytosis are as follows-

Important Similarities

The following are some of the important similarities between endocytosis and exocytosis-

1. Both processes involve membrane remodelling. In endocytosis, the plasma membrane bends and separates. During exocytosis, two lipid membranes join together.
2. Both use membrane-bound vesicles or vesicular structures for movement of materials. The vesicle is formed at plasma membrane in endocytosis, whereas an exocytic vesicle comes from inside of cell.
3. The cytoskeleton and different regulatory proteins are involved in both processes. Actin filaments, microtubules and motor proteins help in movement or positioning of vesicles. Their exact involvement is different between pathways.
4. Endocytosis and exocytosis are parts of cellular membrane trafficking. Both help in maintaining the distribution of receptors, transporters, lipids and other membrane components.
5. Both require cellular energy somewhere in the complete pathway. ATP or GTP is used during vesicle formation, movement, scission, priming, protein-complex disassembly or recycling. It does not mean that every individual membrane step directly hydrolyses ATP.

How the Two Processes Work Together

Endocytosis and exocytosis form a connected membrane cycle. A continuous action of only one process would change the size and composition of plasma membrane.

1. Recovery of added membrane– Exocytosis adds vesicular membrane during secretion. A part of this added membrane is later taken inside by compensatory endocytosis. It prevents continuous enlargement of cell surface.
2. Recycling of synaptic vesicles– In nerve cells, synaptic vesicle membrane and its proteins become exposed at presynaptic membrane after exocytosis. Endocytosis retrieves these components. New functional synaptic vesicles are then formed for another release.
3. Redistribution of surface components– Receptors, transport proteins and lipids are delivered to cell surface by exocytosis. They can later be internalized by endocytosis, sorted in endosomes and again returned or transported for degradation.
4. Maintenance of cell size and composition– The relative rate of both processes controls the plasma-membrane area. Their balance also maintains membrane composition and proper number of cell-surface proteins. The two rates are not necessarily equal at each moment, but become balanced according to the requirement of cell.

Full-Collapse versus Kiss-and-Run Fusion

Full-collapse fusion and kiss-and-run fusion are two modes of exocytotic vesicle fusion. The main difference is in the behaviour of fusion pore and the fate of vesicle membrane.

The differences are as follows-

These two modes are not always completely separated. Intermediate behaviour can also be present. A recent study in rat hippocampal synapses observed a kiss-shrink-run process, in which vesicles opened a small pore, became smaller and many were rapidly recycled. Some vesicles later collapsed into the presynaptic membrane.

Dysregulation and Clinical Relevance of Exocytosis

Defect in exocytosis can decrease secretion or produce uncontrolled release of substances. It may affect nerve communication, hormone secretion, immune defence and blood clotting. The effect depends on the cell and the vesicle which is involved.

The following are some of the important clinical conditions related with exocytosis-

• Botulism– It is caused by neurotoxins produced by Clostridium botulinum. The toxin cleaves particular SNARE proteins required for synaptic vesicle fusion. Acetylcholine is not properly released at the neuromuscular junction. This produces muscle weakness and flaccid paralysis.
• Tetanus– Tetanus toxin is produced by Clostridium tetani. It also attacks the SNARE machinery, especially synaptobrevin or VAMP. The release of inhibitory neurotransmitters such as GABA and glycine is blocked. The motor neurons become overactive and painful muscle spasm or spastic paralysis occurs.
• Diabetes mellitus– Insulin is released from pancreatic beta cells by regulated exocytosis. In type 2 diabetes, insulin-granule docking, priming and fusion may become reduced. A decrease of some exocytotic proteins has also been found. This defect contributes to poor glucose-stimulated insulin secretion, but it is not the only cause of diabetes.
• SNARE-related neurological disorders– Mutations in proteins of synaptic vesicle exocytosis can disturb neurotransmitter release. Pathogenic variants in STXBP1, which forms Munc18-1, affect vesicle docking and fusion. It causes STXBP1 encephalopathy with epilepsy, developmental delay and movement abnormalities. Variants in SNAP25 can also produce developmental and epileptic encephalopathy.
• Familial hemophagocytic lymphohistiocytosis (FHL)– Cytotoxic T cells and natural killer cells destroy target cells by exocytosis of cytotoxic granules. Mutations in UNC13D, STX11 or STXBP2 disturb priming or fusion of these granules. The target cells are not properly killed. Persistent activation of immune cells then produces severe inflammation, fever, cytopenia and organ injury.
• Platelet granule disorders– Activated platelets release ADP, serotonin, adhesive proteins and other materials by granule exocytosis. In Hermansky-Pudlak syndrome, dense granules are deficient. Gray platelet syndrome mainly has deficiency of alpha granules. The platelet release reaction becomes weak and an increased tendency of bleeding may occur.
• Allergy and anaphylaxis– Mast cells contain granules having histamine, proteases and other inflammatory substances. During an IgE-mediated allergic reaction, a large number of these granules may undergo exocytosis. Excessive mediator release causes itching, swelling, bronchoconstriction and fall of blood pressure. A severe systemic reaction is referred to as anaphylaxis.
• Defective plasma-membrane repair– Lysosomal exocytosis has a role in repair of an injured plasma membrane. Dysferlin helps in injury-triggered movement and fusion of lysosomes near the damaged membrane. In dysferlin deficiency, this exocytic repair becomes delayed or reduced. It is associated with muscular dystrophies such as limb-girdle muscular dystrophy type 2B and Miyoshi myopathy.
• Cancer and extracellular-vesicle release– Multivesicular bodies can fuse with plasma membrane and release their internal vesicles as exosomes. Cancer cells may show altered production and release of these vesicles. The vesicles carry proteins, lipids and nucleic acids to other cells. They can take part in tumour communication, immune escape, invasion and treatment resistance. Their use as cancer biomarkers is also being studied.
• Therapeutic use of botulinum toxin– Controlled inhibition of exocytosis is also used during treatment. Botulinum toxin temporarily reduces acetylcholine release from cholinergic nerve endings. It is used in several conditions including focal dystonia, spasticity, hyperhidrosis and overactive bladder. The effect is reversible after recovery of the nerve-terminal exocytotic machinery.

Molecular Proteins Involved in Exocytosis

Different proteins are involved in movement, targeting, docking and fusion of secretory vesicle. Some proteins act before fusion. Other proteins are used during fusion and recycling. The exact protein and its isoform is not same in every cell.

The major molecular proteins involved are as follows-

The SNARE proteins form the main membrane-fusion machinery. Rab proteins and tethering factors bring the vesicle to correct region. Munc proteins prepare it for fusion. In regulated exocytosis, synaptotagmin detects the calcium signal. NSF and α-SNAP act after fusion and recycle the SNARE proteins.

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