CONCLUSIONES Y RECOMENDACIONES.

Regulated secretion is not a universal cellular function, but is carried out by

specialized secretory cells. Material destined for eventual secretion is stored and

concentrated in secretory vesicles or granules. Cells capable o f regulated secretion

only release their contents in response to physiological needs and require specific

signals before they release their cargo. Specialized secretory cells therefore use the

regulated secretory pathway for the rapid release o f neurotransmitters, stored

hormones and enzymes. Mast cells for example degranulate upon cross-linking o f the

high affinity IgE receptor to release a range o f inflammatory mediators. Regulated

secretion is not limited however to the release o f soluble cargo, it is also widely used

for the regulated insertion o f membrane proteins at the cell surface. The appearance o f

the glucose transporter GLUT4 on the surface o f muscle and fat cells is regulated by

insulin (Martin et al., 2000). Therefore many cells are capable o f regulated secretion, although the regulated secretory organelle varies greatly between cell types.

1.3.1 Regulated secretory organelles

The dense-core granules o f neuroendocrine cells begin forming at the trans- Golgi network. It is here that the sorting o f regulated secretory cargo from the

constitutive secretory pathway and from lysosomal enzymes begins. Components

required for the processing o f hormones also are recruited, and the granule begins the

maturation process. Immature secretory granules (ISG) derived from the TGN

undergo maturation by the retrieval o f excess membrane and mis-sorted proteins to

the endosomal pathway via clathrin coated vesicles, leading to condensation o f the

core to form mature secretory granules (Tooze, 1998).

The pathway for biogenesis o f synaptic vesicles is still a matter o f some

debate. One possibility is that immature synaptic vesicles bud directly from the TGN.

Alternatively, they may form through the endocytic pathway after delivery o f

following regulated secretion. Ultrastructural analysis studies do not support a model

whereby synaptic vesicles bud from the TGN (Tsukita and Ishikawa, 1980). The

neuroendocrine cell line, PC 12 has been used extensively to study the biogenesis o f

small synaptic vesicles. In this system, newly synthesized synaptophysin is

transported through the constitutive secretory pathway to the plasma membrane,

before being recruited into synaptic vesicles after multiple rounds o f endocytosis and

exocytosis (Reginer-Vigoroux and Tooze, 1991).

The regulated secretory organelle o f granulocytes is neither a synaptic-like

vesicle nor a dense-core granule. In cells o f the haematopoietic lineage, it is the

lysosome that contains both lysosomal hydrolases and specialized proteins destined

for regulated release upon a stimulus to secrete (table 1.4).

Cell Type Functions Secreted products

Cytotoxic T

lymphocytes and

natural killer cells

Target cell killing Perforin

Granzymes

Eosinophils Defense against parasites Major basic protein

Neurotoxin

Neutrophils Inflammatory response Chemoattractants

Histaminase

Basophils Inflammatory response Histamine

M ast cells Inflammatory response Histamine

Serotonin

Macrophages Phagocytosis

Antigen presentation

Lysosome ‘secretes into

phagosome’

Osteoclasts Bone resorbtion Forms lysosome with bone

Platelets Inflammatory response

Clotting

Clotting factors

Acid and neutral hydrolases

A number o f models have been proposed to describe lysosomal biogenesis.

Originally lysosomes were thought to form directly from the Golgi complex This was

known as the primary lysosome hypothesis and was a long-held view o f lysosome

biogenesis (Bainton, 1981). As our knowledge o f this process increased, this model

appeared no longer valid and other models have been suggested to describe this

pathway. The ‘maturation m odel’ o f lysosomal biogenesis suggests that lysosomes

bud from a late endosomal compartment and non-lysosomal components are removed

from this late endosomal compartment during the maturation process (Roederer et a l, 1990). Alternatively, the ‘vesicle shuttle m odel’ suggests that small vesicles bud from

late endosomes for the selective delivery o f lysosomal components destined for a pre­

existing lysosome. Lysosomes therefore grow by expansion o f pre-existing membrane

and division through a fission process. However, no evidence exists to date supporting

this hypothesis. Finally, a third model for the biogenesis o f lysosomes, termed the

‘kiss and run’ hypothesis (Storrie and Desjardins, 1996) has been suggested. Here,

pre-existing lysosomes undergo repeated, transient fusion and fission processes with

late endosomes. This pathway therefore can be used to acquire newly synthesized molecules and components o f the endocytic pathway. A modification to this model

has also been suggested involving direct and complete fusion between endosomes and

lysosomes with subsequent recovery o f lysosomes for re-use (Griffiths, 1996a; Luzio et al., 2000). Clearly, the lysosomal biogenesis pathway is still far from understood. Advances in the field o f molecular cell biology and the use o f animal models

defective in lysosomal biogenesis and function such as the mouse coat colour

mutants, with their defects in melanosome biogenesis and function should increase

our knowledge o f this pathway.

It is not clear if cells o f the haematopoietic lineage possess specialized

lysosomes, or if all cells can secrete lysosomal proteins in response to appropriate

signals. Evidence from studies using rat kidney (NRK) fibroblasts demonstrates that

elevation o f intracellular free calcium with calcium ionophore results in the fusion o f

lysosomes with the plasm a membrane (Rodriguez et a l, 1997). This suggests that fusion o f lysosomes with the plasm a membrane in response to calcium may be a

function common to all cells. Evidence that haematopoietic cells possess specialized

‘secretory lysosomes’ comes primarily from studies o f patients with the genetic defect

extensive hypopigmentation o f the skin, hair and eyes. Melanocytes containing giant

melanosomes unable to transfer to the surrounding kératinocytes seem to be

responsible for the decreased pigmentation in CHS patients exhibiting albinism.

These mutants have been widely used as a model system to study lysosome function

in haematopoietic cells. Although all cell types examined from CHS patients

exhibited giant lysosomes, only haematopoietic cells and melanocytes demonstrated a

functional defect, with an inability to secrete their lysosomes. This suggests that cells

o f this lineage may share a common mechanism o f secretion distinct from other

regulated secretory cell types. The gene found to be mutated in CHS has recently been

cloned (Pérou et a l, 1996; Barbosa et a l, 1996). More recently the CHS gene product has been demonstrated to be a 400kDa cytosolic protein, possibly involved in

lysosome fission (Pérou et a l, 1997), although it has also been implicated in sorting resident endosomal proteins into late multivesicular endosomes by a microtubule

dependant mechanism (Faigle et a l, 1998). Neurobeachin, a neuronal homologue o f the CHS gene has recently been identified. Like the CHS gene product it is implicated

in membrane trafficking. It also has a high affinity binding site for the type II

regulatory subunit o f protein kinase A (Wang et a l, 2000). As a clearer understanding o f the function o f the CHS gene product and other proteins involved in this pathway is

gained, our insights into the formation o f the secretory lysosome, and the regulated

secretory pathway o f these specialized secretory cells should be furthered.